Syntaxin-3 Regulates Biphasic Glucose Stimulated Insulin Secretion in the Pancreatic Beta Cell

by

Ellen Koo

A thesis submitted in conformity with the requirements for the degree of Masters of Science

Graduate Department of Physiology

University of Toronto

© Copyright by Ellen Koo (2010)

Syntaxin-3 Regulates Biphasic Glucose Stimulated Insulin Secretion in the Pancreatic Beta Cell

Ellen Koo

Masters of Science (2010)

Department of Physiology

University of Toronto

Abstract

The molecular basis of exocytosis of insulin granules during biphasic glucose stimulated

insulin secretion (GSIS) of pancreatic beta cells remains unclear. Of the exocytotic Syntaxin

(Syn) family of SNARE proteins, Syn-1A has been postulated to mediate exocytosis of docked

insulin granules during first phase GSIS, while Syn-4 facilitates both phases of GSIS. Syn-3 is

also abundant in islet beta cells, but its function in GSIS is unknown. Our study aims to

investigate the role of Syn-3 in biphasic insulin secretion and how it regulates the recruitment to

plasma membrane and/or exocytotic fusion of insulin granules. Confocal microscopy showed

endogenous Syn-3 (and exogenously overexpressed Syn-3 WT-EGFP) to be localized to insulin

granules. We first examined the function of endogenous Syn-3 by down-regulating its

expression employing siRNA and lenti-shRNA viruses, the latter to establish a stable knockdown

INS-1 cell line; which impaired GSIS. Total internal reflection fluorescence microscopy

(TIRFM) of Syn-3 depleted INS-1 cells showed no change in the number of docked insulin

granules and preservation of fusion competence of previously docked granules – which are

functions previously attributed to Syn-1A. Remarkably, Syn-3 depletion caused marked

reduction in the number of newcomer granules and their subsequent exocytotic fusion, which

encompassed both first and second phases of GSIS. We then examined the effects of

ii

overexpressing Syn-3 WT-EGFP, which enhanced biphasic insulin secretion in mice islets.

Since open conformation Syn-1A was previously reported to enhance Syn-1A actions by

promoting exocytotic SNARE complex formation, we constructed open form Syn-3. Exogenous

open form Syn-3 had no effect on secretion as it is retained in the cytosol, unable to be trafficked

to insulin granules. Taken together, we conclude that Syn-3 in insulin granules functions to

facilitate (or mediate) mobilization of newcomer insulin granules to the plasma membrane, to

contribute to both first and second phases of GSIS in pancreatic beta cells.

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Acknowledgements I would like to thank my supervisor Dr. Herbert Gaisano, for giving me the opportunity to further

my learning in the field of research and for showing me patience and understanding during the

completion of this project.

I also wish to thank my supervisory committee members, Dr. Shuzo Sugita, Dr. Allen Volchuk

and Dr. Michael Wheeler, for sharing their knowledge and providing direction in my project.

This thesis would have not been possible without the support and expertise of my fellow lab

members. I wish to thank Dr. Dan Zhu, for the contributions to the TIRFM analysis of my

project. I especially want to thank Dr. Edwin Kwan, for teaching me the techniques used in my

experiments and the constructive feedback on my thesis. I also wish to thank Dr. Youhou Kang

and Huanli Xie, for the invaluable assistance that was always offered without hesitation.

Lastly, I would like to thank God, my parents, sister and friends for their love and

encouragement through the duration of my graduate studies.

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Table of Contents Abstract ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ii Acknowledgements ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... iv Table of Contents ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... v List of Figures ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... viii Chapter 1: Introduction ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 1

1.1 Exocytosis in Secretory Cells ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 1

1.1.1 Primary Exocytosis ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 1

1.1.2 Granule-Granule Fusion Underlying Sequential and Compound

Exocytosis ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 2

1.2 The Pancreatic Beta Cell as a Model of Exocytosis ... ... ... ... ... ... ... ... ... ... ... 3

1.3 Physiology of Biphasic Insulin Secretion ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 6

1.4 The SNARE Family and SNARE Hypothesis ... ... ... ... ... ... ... ... ... ... ... ... ... ... 8

1.4.1 The Syntaxin Family ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 11

1.4.2 Syntaxin-1 Regulation of Insulin Secretion in the Beta Cell ... ... ... ... 13

1.4.3 Syntaxin-4 Regulation of Insulin Secretion in the Beta Cell ... ... ... ... 15

1.5 SNAREs in the Pathophysiology of Type-2 Diabetes ... ... ... ... ... ... ... ... ... ... 17

1.6 The SM Protein Family and Interactions with SNAREs ... ... ... ... ... ... ... ... ... 18

1.7 Cellular Functions of Syntaxin-3 in Various Tissues ... ... ... ... ... ... ... ... ... ... ... 21

1.8 Rationale and Hypothesis – Syntaxin-3 Recruitment of Newcomer Insulin

Granules during Biphasic Glucose Stimulated Insulin Secretion ... ... ... ... ... ... 22

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Chapter 2: Research Design and Methods ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 24 2.1 INS-1 (832/13) Cell Culture ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 24

2.2 Immunoblotting ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 24

2.3 siRNA Transfection of INS-1 Cells ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 25

2.4 Static Insulin Secretion Assay ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 25

2.5 Lentivirus Transduction and Establishment of New Stable Cell Lines ... ... ... ... 26

2.6 Total Internal Refraction Fluorescence Microscopy ... ... ... ... ... ... ... ... ... ... ... ... 26

2.7 Cell Preparation for Confocal Immunofluorescence Microscopy ... ... ... ... ... ... ... 31

2.8 Adenovirus Production and Purification ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 31

2.9 Adenoviral Transduction of INS-1 Cells ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 33

2.10 Isolation of Mouse Pancreatic Islets and Beta Cells ... ... ... ... ... ... ... ... ... ... ... 33

2.11 Adenoviral Transduction of Mouse Islets ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 34

2.12 Mouse Islet Perifusion ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 34

2.13 Statistics ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 34

Chapter 3: Results ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 35

3.1 Syntaxin-3 Expression in Pancreatic Islets and Colocalization with Insulin

Granules ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 35

3.2 RNAi-mediated Depletion of Syntaxin-3 Impaired Glucose Stimulated Insulin

Secretion in INS-1 Cells ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 37

3.3 Diminished Glucose Stimulated Insulin Secretion in Stable Syntaxin-3

Knockdown ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 39

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3.4 Syntaxin-3 Depletion Reduced Newcomer Granule Mobilization Underlying

Biphasic Insulin Secretion ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 43

3.5 Syntaxin-3 Overexpression Potentiated Glucose Stimulated Insulin Secretion in

Mouse Islets ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 49

3.6 Overexpression of Syntaxin-3 (in Wild Type Conformation) Enhanced Glucose

Stimulated Insulin Secretion in INS-1 Cells ... ... ... ... ... ... ... ... ... ... ... ... ... ... 55

3.7 Mislocalization of Open-Form Syntaxin-3 Following Overexpression in

INS-1 Cells ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 57

Chapter 4: Discussion

4.1 Syntaxin-3 Facilitates Exocytosis of Newcomer Granules during Biphasic

Glucose Stimulated Insulin Secretion ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 59

4.2 Experimental Limitations ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 64

4.3 Future Directions ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 65

4.4 Physiological Relevance of Syntaxin-3 Function in Glucose Stimulated Insulin

Secretion ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 68

References ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 70

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List of Figures

Figure 1 Glucose stimulated insulin secretion by the pancreatic beta cell ... ... ... ... ... 5

Figure 2 Biphasic pattern of glucose stimulated insulin secretion in the pancreatic

beta cell ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 7

Figure 3 SNARE complex formation and the SNARE hypothesis ... ... ... ... ... ... ... ... ... ... 10

Figure 4 Syntaxin protein conformation ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 12

Figure 5 Schematic drawing of the evanescent field by TIRFM ... ... ... ... ... ... ... ... ... ... 29

Figure 6 TIRFM of insulin granules labelled with Syncollin-pHluorin ... ... ... ... ... ... ... ... 30

Figure 7 Rat Syntaxin-1A and Syntaxin-3 protein sequence alignment ... ... ... ... ... ... ... 32

Figure 8 Endogenous expression of Syntaxin-3 in mouse pancreatic islets and

beta cells ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 36

Figure 9 RNAi-mediated depletion of Syntaxin-3 impaired glucose stimulated insulin

secretion ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 38

Figure 10 Stable Syntaxin-3 knockdown INS-1 clones established using lenti-shRNA ... ... 40

Figure 11 Diminished glucose stimulated insulin secretion in stable Syntaxin-3 knockdown

cells ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 41

Figure 12 Immunoblot analyses examining exocytotic protein expression ... ... ... ... ... ... 42

Figure 13 Syntaxin-3 depletion reduced the total sum of fusion events underlying biphasic

insulin secretion ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 45

Figure 14 TIRFM of INS-1 cells expressing NPY-EGFP insulin vesicle cargo protein

differentiated between pre-docked and newcomer granule fusion modes ... ... ... 46

Figure 15 Syntaxin-3 depletion did not affect the vesicle density at the plasma membrane

prior to stimulation ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 47

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Figure 16 Syntaxin-3 depletion reduced newcomer granule mobilization underlying

biphasic insulin secretion ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 48

Figure 17 Syntaxin-3 overexpression and transduction efficiency in mouse islets ... ... 51

Figure 18 Confocal microscopy of whole mouse islet core ... ... ... ... ... ... ... ... ... ... ... 52

Figure 19 Syntaxin-3 overexpression potentiates glucose stimulated insulin

secretion in mouse islets ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 53

Figure 20 Cellular insulin content of mouse islets ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 54

Figure 21 Overexpression of Syntaxin-3 (wild type) enhanced glucose stimulated insulin

secretion in INS-1 cells ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 56

Figure 22 Mislocalization of open-form Syntaxin-3 following overexpression in

INS-1 cells ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 58

Chapter 1

Introduction

1.1 Exocytosis in Secretory Cells

Exocytosis is the process by which vesicles traffic towards the cell periphery and fuse

with the plasma membrane. The principle steps of exocytosis can be further broken down into

vesicle trafficking, tethering, docking, priming and fusion. Membrane traffic initiates with

vesicle budding from a precursor compartment and proceeds with mobilization of granules to

future release sites. Tethering and docking of vesicles then occurs in plasma membrane regions

that are enriched in clusters of exocytotic proteins. To become fusion competent, granules must

be primed through a number of reactions which are ATP, Ca2+ and temperature dependent. The

final exocytotic event utilizes a narrow fusion pore that connects the inside of the granule to the

extracellular environment. Upon vesicle fusion, soluble particles are released into the

extracellular fluid, while lipids and integral membrane proteins are incorporated into the plasma

membrane and displayed at the cell surface. Since the vesicle membrane fuses with the plasma

membrane of the cell, this mechanism also has a role in maintaining cell integrity by restoring

membrane lost during endocytosis.

1.1.1 Primary Exocytosis

Primary exocytosis is characterized by the fusion of the secretory granule and the plasma

membrane. In the classical view of exocytosis, vesicles fully collapse into the membrane and

1

these components are later retrieved through endocytosis. However, electron microscopy of

exocytotic events has shown that synaptic vesicles may not necessarily undergo complete fusion,

but can briefly interact with the membrane in a “kiss-and-run” manner (Fesce et al. 1994). Kiss-

and-run exocytosis, also termed “reversible” fusion, is characterized by the ability of the fusion

pore to rapidly open and close as long as a certain diameter is not exceeded (MacDonald et al.

2005). Kiss-and-run exocytosis is also a mechanism for differential release of insulin since the

withdrawal back into the cytosol prevents the complete discharge of granule contents. In

addition, the transient nature of reversible fusion occurs without adding components to the

plasma membrane, eliminating the need for subsequent membrane retrieval. In recent reports, it

has been shown that kiss-and-run exocytosis may be the more prevalent type of fusion event

accounting for up to ~75% of exocytotic events in islet beta cells, which although result in

incomplete or no emptying of insulin cargo, would provide effective release of other smaller

resident granule cargo such as nucleotides (Obermuller et al. 2005).

1.1.2 Granule-Granule Fusion Underlying Sequential and Compound Exocytosis

Secondary exocytosis occurs upon fusion of granules with other granules and this can be

further categorized into sequential and compound exocytosis. Compound exocytosis

encompasses the two distinct processes of sequential exocytosis and multigranular exocytosis

(Alvarez de Toledo, Fernandez 1990). In sequential exocytosis, vesicles fuse with other vesicles

that have already fused with the plasma membrane. This process has been observed in glucose

stimulated islet cells, which show the orderly fusion of oncoming secretory granules with

granules already fused with the plasma membrane (Kwan et al. 2007, Kwan, Gaisano 2005). For

2

simplicity, multigranular exocytosis is termed compound exocytosis as used in the context of this

report. Compound exocytosis occurs when multiple vesicles have already undergone fusion in

the cytosol before surfacing to and fusing with the plasma membrane. This phenomenon is also

termed homotypic fusion and may allow for the unloading of a greater amount of cargo

following fusion of the granule with the plasma membrane.

1.2 The Pancreatic Beta Cell as a Model of Exocytosis

The storage and release of insulin by islet beta cells plays an essential role in glucose

homeostasis, lipid and protein metabolism, brain function and cell survival. Remarkably, the

release of this hormone is not constitutive, but highly controlled as ~99% of insulin secretion

follows a regulated pathway (Rhodes, Halban 1987). The beta cell is electrically excitable and

couples changes in extracellular glucose levels to insulin exocytosis. As plasma glucose rises,

GLUT-2 receptors on the cell surface transport glucose across the plasma membrane and into the

cytosol. As glucose is metabolized and ATP levels increase, the rise in the ATP/ADP ratio

closes KATP channels, resulting in cell depolarization. In response, voltage-dependent calcium

channels open and the Ca2+ influx initiates the beginning of biphasic insulin exocytosis. As

insulin promotes glucose uptake by the target organs, plasma glucose falls, reversing this

pathway to end the secretory process. Thus, glucose homeostasis is under feedback control of

insulin via changes in beta cell metabolism, electrical activity and KATP channel closure (Figure

1).

3

Newly synthesized insulin is packaged into dense core vesicles and each beta cell

contains approximately 10 000 granules. The first phase of secretion is attributed to a readily-

releasable pool (RRP) of insulin granules located directly beneath the plasma membrane

(Rorsman, Renstrom 2003). These granules are secreted immediately as they are pre-docked and

primed for release. Ultrastructural studies have revealed that ~50-200 granules are docked at the

plasma membrane while another ~1500 granules are situated within 0.2µm from the cell surface

(Olofsson et al. 2002). The vast majority of insulin granules are located within storage pools in

the cytosol and second phase release constitutes the recruitment of these vesicles to refill the

RRP. This replenishment process has been reported to occur at a rate of 5-40 granules per cell

per minute (Olofsson et al. 2002). Since granule priming is necessary for release competency,

the second phase of secretion is the rate-limiting step of insulin exocytosis (Easom 2000).

4

Figure 1. Glucose stimulated insulin secretion by the pancreatic beta cell. Following the uptake of glucose into the pancreatic beta cell through GLUT-2 receptors, glucose is metabolized through glycolysis to increase intracellular ATP levels. The increased ATP/ADP ratio induces closing of KATP channels, leading to cell depolarization and subsequent calcium influx. Increased calcium concentrations lead to a series of intracellular signals which triggers the beta cell exocytotic machinery to secrete insulin. Adapted with permission from J Transl Med. 2007;5 (Copyright 2007 Ren et al; licensee BioMed Central Ltd.)

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1.3 Physiology of Biphasic Insulin Secretion

Insulin release was first reported to secrete in a biphasic manner in 1968 (Curry, Bennett

& Grodsky 1968), and since then has been further characterized as a ‘storage-limited’ model of

exocytosis (Henquin et al. 2002). Under current concepts, biphasic insulin secretion corresponds

to the exocytosis of spatially or functionally distinct pools of granules as shown in Figure 2. The

first phase of insulin release develops rapidly and peaks within 5-10 minutes after stimulation.

However, this initial surge is relatively transient and shortly after, secretion decreases to a level

that is higher than basal release. Since the small pool of RRP granules is depleted within

minutes of stimulation, the first phase of insulin secretion also includes the recruitment and

release of newcomer insulin granules. Therefore, first phase secretion constitutes both the

secretion of pre-docked granules and the initiation of RRP replenishment.

While first phase release can be induced by KCl and glucose, the second phase of insulin

secretion occurs exclusively following stimulation by nutrients (Gembal, Gilon & Henquin

1992). Also in contrast to first phase, second phase release develops slowly, but can be sustained

over several hours during elevated glucose conditions (Curry, Bennett & Grodsky 1968). During

second phase secretion, insulin vesicles are recruited from the reserve pool of granules in the

cytosol to replenish the RRP near the plasma membrane. Upon arrival at the RRP, these

newcomer granules are primed and subsequently docked for exocytotic release. The second

phase response holds greater quantitative importance since only 1% of total insulin granules are

released during first phase secretion, allowing for the emptying of a greater amount of insulin

cargo (Ohara-Imaizumi et al. 2007).

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Figure 2. Biphasic pattern of glucose stimulated insulin secretion in the pancreatic beta cell. The biphasic pattern of GSIS is attributed to the exocytosis of two pools of insulin granules. The release of the RRP (readily releasable pool) granules occur immediately following glucose stimulation and accounts for the transient first phase peak of GSIS. The second more sustained phase of secretion corresponds to the mobilization of insulin granules from the reserve pool to refill the RRP and priming of these granules to become fusion competent. Adapted with permission from J Transl Med. 2007;5 (Copyright 2007 Ren et al; licensee BioMed Central Ltd.)

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1.4 The SNARE Family and SNARE Hypothesis

SNAREs are a superfamily of proteins that function in membrane fusion events of the

secretory pathway. SNARE proteins are receptors for SNAP (soluble NSF attachment protein)

and NSF (N-ethylmaleimide-sensitive factor), which are required for transport vesicle fusion in a

cell-free system (Sollner et al. 1993). Although variable in size and structure, these proteins

share a common 60-70 residue “SNARE motif” sequence containing heptad repeats (Ungar,

Hughson 2003). With the exception of synaptosomal associated protein SNAREs which contain

two motifs, the majority of SNARE proteins are anchored to the membrane at the C-terminal and

contain a single SNARE motif. Each SNARE motif is further classified under the structural

category of R-, Qa, Qb and Qc, and all functional SNARE complexes contain one of each type of

motif (Fasshauer et al. 1998). The Q/R classification of SNAREs refers to the conserved amino

acid residue at the zero layer of the SNARE four-helical bundle (see below). R-SNAREs

contribute an arginine (R) at this position and are usually vesicle-associated (v-SNAREs). Q-

SNAREs are target membrane-associated (t-SNAREs) and contribute a glutamine (Q) in the zero

ionic layer of the assembled SNARE complex. Q-SNAREs are further classified as Qa for the

Syntaxin subfamily, or Qb and Qc for the SNAP-25 N-terminal and C-terminal subfamily

respectively.

The conserved SNARE motif is essential for the formation of a four-helix bundle known

as the trans-SNARE complex or SNAREpin shown in Figure 3. The four-helix bundle is

composed of a combination of four SNARE motifs contributed by three or four different

SNAREs. The four helices interact across 16 layers and the middle of the bundle is defined as

8

the zero layer which dictates the Q/R classification of SNAREs mentioned above (Fasshauer et

al. 1998). The SNARE complex constitutes the core machinery required for each step of

membrane fusion and a specific set of four SNARE motifs corresponds to distinct exocytotic

events. SNAREs are proposed to catalyze membrane fusion in a process that involves the t-

SNARE helices and the v-SNARE helix interacting sequentially in layers to form the trans-

complex. During membrane fusion, the SNARE complex converts from “trans” to “cis” and the

SNARE motifs align in a manner resembling a molecular zipper. As the helices zipper up from

the N-termini towards the C-termini, the gap between the vesicle and plasma membrane closes.

This generates an inward force which drives fusion by pulling the bilayers together. The energy

released during this process is used to overcome the fusion barrier and after complete zippering

of the SNARE motifs, a lower energy stable cis-complex results (Hua, Scheller 2001).

The SNARE hypothesis proposes that SNARE proteins constitute the core machinery

essential for membrane fusion. Simply put, each membrane participating in fusion must contain

at least one anchored SNARE. Under the hypothesis, the interactions of distinct sets of v-

SNAREs and t-SNAREs regulate membrane fusion by ensuring vesicle specificity, targeting and

fusion (Sollner et al. 1993). Since only a small combination of SNAREs are fusogenic and

correspond to specific transport processes, SNAREs are considered one of the major components

dictating the specificity of membrane fusion. Interestingly, SNARE interactions in vitro have

shown considerable promiscuity during fusion events (Grote, Novick 1999, Bajohrs et al. 2005),

suggesting the existence of additional control mechanisms involved in exocytosis.

9

Figure 3. SNARE complex formation and the SNARE hypothesis. Vesicle associated SNAREs interact with target membrane associated SNAREs to assemble a four-helix SNARE complex essential to driving membrane fusion. Adapted by permission from Macmillan Publishers Ltd: Nature Reviews Molecular Cell Biology. 2001;2, 98-106. (Copyright 2001)

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1.4.1 The Syntaxin Family

Syntaxins are a large mammalian family of t-SNARE proteins which are specialized for

regulating exocytosis. Syn-1 was first discovered in the plasma membrane of neurons and since

then, numerous isoforms have been found in a variety of cell types (Bennett et al. 1993). The

majority of Syntaxins are transmembrane anchored proteins and are localized on cell

compartments such as the ER, Golgi and endosomes (Teng, Wang & Tang 2001). Syntaxin is

anchored in lipid membranes at the hydrophobic C-terminal, while the N-terminal remains in the

cytosol. The SNARE motif is on the C-terminal and is highly conserved between all Syntaxin

proteins. Located at the N-terminal is an alpha-helical region, termed the Habc domain, which is

connected to the SNARE motif via a long flexible linker (or hinge) region. In its wild-type state,

Syntaxin can rapidly switch between an “open” or “closed” protein conformation (Margittai et al.

2003) as shown in Figure 4. When not bound in the SNARE complex, Syntaxin interacts

intramolecularly due to the folding of the Habc domain to mask the C-terminal SNARE motif. In

this conformation, Syntaxin cannot bind to other SNAREs and is considered “closed.” When

assembling into the SNARE complex, Syntaxin unfolds into the “open” conformation, exposing

the SNARE motif to participate in the four-helix bundle formation essential for fusion (Gerber et

al. 2008, Dulubova et al. 1999). Of the numerous syntaxin isoforms, Syn-1,-2,-3 and -4 are

implicated in regulating insulin exocytosis in pancreatic beta cells (Wheeler et al. 1996). While

the function of Syn-2 and -3 remain unclear, there has been considerable progress in defining the

roles of Syn-1 and -4 during insulin secretion.

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Figure 4. Syntaxin protein conformation. Syntaxin can adopt an open conformation in which the SNARE motif is available to participate in SNARE complex formation which is essential for membrane fusion. However, when the Habc domain folds over to mask the SNARE motif, Syntaxin cannot bind to other SNAREs and is considered “closed.” Adapted with permission from Osborn and Jorgensen 2007, University of Utah.

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1.4.2 Syntaxin-1 Regulation of Insulin Secretion in the Beta Cell

In the neuronal presynaptic membrane, Syntaxin-1 (Syn-1), SNAP-25 and vesicle-

associated membrane protein-2 (VAMP-2, synaptobrevin) form a SNARE complex which

functions in synaptic vesicle fusion (Rothman 2002). The crucial role of these exocytotic

proteins was revealed when synaptic vesicle fusion was impaired following SNARE targeted

proteolysis by neurotoxins (Boyd et al. 1995, Jahn, Niemann 1994). Since these SNAREs are

also expressed in the pancreatic beta cell, the trimeric complex was proposed to mediate the

secretion of insulin granules (Gerber, Sudhof 2002). Consistent with this hypothesis, ~400

aggregates of Syn-1/SNAP-25 complexes were seen in the mouse beta cell (Vikman et al. 2006),

which is remarkably similar to the number of docked granules reported in ultrastructural studies

(Olofsson et al. 2002). Perifusion of Syn-1A knockout mice islets showed a blunted first phase

secretion, supporting an essential role for Syn-1A in the docking and fusion of insulin granules in

the RRP of pancreatic beta cells (Ohara-Imaizumi et al. 2007).

Aside from regulating granule fusion during first phase release, Syn-1A interacts with

three important ion channels to mediate beta cell excitability and insulin exocytosis (Leung et al.

2003, Neshatian et al. 2007, Ahmed et al. 2007). Firstly, Syn-1A has been shown to regulate

KATP channel closure which triggers cell depolarization and subsequent insulin exocytosis (Cui et

al. 2004). By binding to the sulfonylurea receptor-1 (SUR1) regulatory component of the KATP

channel, Syn-1A effectively inhibits current activity (Pasyk et al. 2004). This was demonstrated

by an increase in KATP channel activity following botulinum toxin C1 mediated cleavage of Syn-

1A in HIT beta cells (Pasyk et al. 2004). It was also determined that the N-terminal H3 domain

13

mediated channel inhibition, revealing the functional importance of Syn-1A in open

conformation. Since KATP channel inhibition initiates the cascade of events which ultimately

culminates in secretion of insulin, Syn-1A regulation is crucial to beta cell exocytosis.

Another target of Syn-1A is Kv2.1, which is the major delayed rectifier K+ channel in

beta cell repolarization (MacDonald, Wheeler 2003). Syn-1A has been reported to inhibit Kv2.1

function by interacting with the C-terminus of the channel (Leung et al. 2003). Overexpression

of Syn-1A impaired trafficking of Kv2.1 channels to the plasma membrane and modified gating

properties by delaying channel activation (Leung et al. 2003). Structure-function studies also

showed Syn-1A and Kv2.1 binding inhibited channel activity and decreased the current

amplitude (Leung et al. 2003). Further investigation revealed Syn-1A also reduced Kv2.1

channel availability upon steady-state depolarization (Leung et al. 2005). Interestingly, both

closed and open form Syn-1A appears to inhibit channel function although the open

conformation is more potent in blocking current activity (Leung et al. 2005). This inhibition by

Syn-1A can significantly alter exocytosis as Kv2.1 blockage impairs repolarization which

subsequently enhances Ca2+ influx and sustains insulin secretion (MacDonald, Wheeler 2003).

Kv2.1 channels have also been shown to interact with Syn-1 bound to SNAP-25 (Tsuk et al.

2005). This interaction was suggested to stabilize the t-SNARE complex at the plasma

membrane and determine the rate of association between Syn-1/SNAP-25 and VAMP-2 on the

insulin granule. Since formation of the full SNARE complex resulted in complete dissociation

from the channel, Kv2.1 appears to stabilize the transition state of the t-SNARE complex prior to

fusion (Tsuk et al. 2008). In this context, it is likely that Syn-1/SNAP-25/Kv2.1 association acts

14

as a scaffold for the docking of insulin granules in preparation for SNARE complex assembly

and exocytotic fusion.

Lastly, Syn-1A interacts with L-type voltage dependent calcium channels (VDCC) which

control cellular Ca2+ levels during exocytosis in beta cells of rats, mice and humans (Sheng et al.

1996, Schulla et al. 2003, Yang, Berggren 2006). L-type VDCC is critical to both phases of

secretion since the Ca2+ influx triggers KATP channel dependent insulin exocytosis, while

intracellular Ca2+ signaling is required for RRP replenishment. Syn-1A has been shown to

regulate L-type VDCC by maintaining channel activity and preventing rundown (Yang et al.

1999). However, overexpression of Syn-1A also reduced L-type VDCC activity and exocytosis

in beta cells and insulinoma cell lines (Kang et al. 2002). Sensitivity to Syn-1A expression may

be linked to the assembly of SNARE complexes which occurs in the vicinity of L-type VDCC.

This provides advantages as SNARE complex formation in these calcium microdomains

simultaneously allows for a quick response and fast feedback in controlling the influx of Ca2+

(Leung et al. 2007).

1.4.3 Syntaxin-4 Regulation of Insulin Secretion in the Beta Cell

Syn-4 plays an important role in coupling pancreatic insulin secretion to the uptake of

glucose into skeletal muscle and adipose tissue (Hou, Pessin 2007). Together with SNAP-23 and

VAMP-2, Syn-4 regulates the translocation of GLUT-4 receptors to the plasma membrane of

adipocytes and muscle cells. Various reports have also demonstrated that Syn-4 is implicated in

15

insulin granule exocytosis. A study overexpressing Synip, a Syn-4 binding partner and

sequestration agent, led to impaired insulin release in beta-HC-9 cells (Saito et al. 2003).

Perifusion of Syn-4 (-/+) mice islets showed reduced biphasic glucose stimulated insulin

secretion (Spurlin, Thurmond 2006), while Syn-4 overexpression in mouse beta cells amplified

the first and second phase of release (Spurlin, Thurmond 2006). Syn-4 (-/+) mice were also

found to be insulin resistant and glucose intolerant 4-6 months after birth. However, islets

collected from these mice at the ages of 2-3 months demonstrated normal glucose tolerance

(Spurlin, Thurmond 2006). These data indicate that secretory defects may precede insulin

resistance and further support a positive role for Syn-4 in exocytotic release.

The precise mechanism by which Syn-4 mediates insulin exocytosis remains unclear.

Although Syn-4 association with VAMP-2 may be involved (Oh, Thurmond 2009), the

significance of this interaction still needs to be elucidated. Not surprisingly, the protein

conformation of Syn-4 may specify between a positive or negative effect on secretion. Recently,

it was reported that Syn-4 in closed conformation tethers F-actin to the plasma membrane and

prevents granule docking. The corresponding decrease in the number of docked granules

implicated closed form Syn-4 in the negative regulation of exocytosis. Further support for this

role was seen when glucose induced conversion of Syn-4 into open conformation disrupted

cytoskeleton binding, and subsequently enhanced insulin secretion (Jewell et al. 2008).

16

1.5 SNAREs in the Pathophysiology of Type-2 Diabetes

Impairments in biphasic secretion are an early indicator of beta cell dysfunction and

considered a major contributor to type-2 diabetes mellitus. Secretory defects from islets of type-

2 diabetic patients and rodent models are strongly correlated with reduced expression of SNARE

proteins Syn-1A, SNAP-25 and VAMP-2 (Chan et al. 1999, Ostenson et al. 2006). Remarkably,

when Syn-1A and SNAP-25 levels were restored with adenoviral delivery vectors, the secretory

deficiency was reversed (Nagamatsu et al. 1999). Decreased expression of exocytotic SNAREs

reduces the number of fusogenic SNARE complexes and consequently inhibits insulin release.

SNAREs also regulate various ion channels mediating insulin release and altered channel

function negatively impacts the secretory process. For example, changes in cellular Ca2+ influx

may affect the nature of fusion events as high calcium concentrations were reported to shift the

mechanism of secretion from kiss-and-run to complete fusion (Elhamdani, Azizi & Artalejo

2006). It is therefore speculated that the opposite effect may occur in diabetes, and the

likelihood of kiss-and-run fusion events is increased (Eliasson et al. 2008). Interestingly, a study

supporting this theory showed that rat islets exposed to 48 hours of high glucose conditions

exhibited a higher ratio of kiss-and-run events, while only 5% of exocytotic events proceeded to

full fusion (Tsuboi et al. 2006). This suggests that impaired insulin release may be related to

incomplete emptying of insulin cargo during exocytosis induced by elevated glucose levels.

Adding to the complexity of the disease, certain ion channel impairments may not exhibit as

strong of a correlation to secretory defects in diabetes. This was the case when glibenclamide, a

pharmacological KATP channel inhibitor, was used to stimulate insulin secretion in diabetic rat

islets. Following drug administration, secretion by diabetic islets was identical to the results of

17

glucose stimulated insulin release (Tsuboi et al. 2006). Thus, defects in the exocytotic

machinery may be severe enough to result in the manifestation of type-2 diabetes, independent of

KATP channel activity.

1.6 The SM Protein Family and Interactions with SNAREs

The Sec1/Munc18 (SM) proteins are a family of cytosolic proteins which have been

isolated in various species of mammals and yeast (Gallwitz, Jahn 2003). SM proteins were first

discovered when examining yeast and C. elegans mutants exhibiting impaired membrane

trafficking and secretion. Genetic deletion of specific SM proteins resulted in profound secretory

defects, further indicating the requirement of SM proteins in membrane fusion. SM proteins

function to regulate the assembly of the SNARE complex and interact with t-SNAREs through

association with other proteins. This association may be stronger than anticipated as deletion of

a SM protein also reduced the expression of its respective SNARE binding partner (Gallwitz,

Jahn 2003). The structure of SM proteins resembles a clasp and contains a central cleft that may

or may not interact with SNAREs. This results in multiple binding modes, which may account

for differential effects in regulating SNARE function. In the most common mode, a SNARE

protein binds to a small groove on the SM protein surface, forming a complex which functions in

trafficking steps involving endosomes, ER and Golgi network.

Numerous studies demonstrated the critical role of SM proteins in SNARE mediated

exocytosis such as impaired neurotransmitter release by neurons of Munc18-a knockout mice

18

(Verhage et al. 2000). Munc18-a (or Munc18-1), expressed primarily in neurons and

neuroendocrine cells, has been known to interact with Syn-1,-2 and -3. The binding mode

between Munc18-a and Syn-1, however, is the most unique and well characterized. During this

interaction, closed form Syn-1A is inserted into the central cleft of Munc18-a (Sudhof, Rothman

2009). This binding prevents unfolding of Syntaxin into open conformation and inactivates the

SNARE function of the protein. Although this led to initial suggestions of Munc18-a as a

negative regulator of membrane fusion, later studies revealed Munc18-a overexpression had no

observable effect on exocytosis (Gallwitz, Jahn 2003). It is therefore more likely that Munc18

binding functions to traffic SNAREs towards target membrane compartments to regulate

exocytosis (Arunachalam et al. 2008). In contrast to the aforementioned inhibitory effect of

Munc18, later theories proposed Munc18-a to promote exocytosis. Other hypothesis suggested

SM proteins may be involved in SNARE complex formation and proofreading by differentiating

between cognate and noncognate partners (Peng, Gallwitz 2002). There is in fact, substantial

evidence for these theories as Munc18-a was found to interact with binding partners that promote

assembly of SNARE complexes. Munc18-a also stabilized a half-closed conformation of

Syntaxin which participated in SNARE complex assembly (Zilly et al. 2006) and also activated

SNARE-mediated membrane fusion in a reconstituted liposome system (Shen et al. 2007). In the

latter case, Munc18-a was observed to associate directly with the assembled SNARE complex

(Dulubova et al. 2007).

Apart from Munc18-a, mammalian homologue Munc18-c (Munc18-3) has also been

implicated in regulating vesicle fusion at the plasma membrane. Munc18-c is expressed

ubiquitously and can bind to Syn-2 and -4 in islets and beta cell lines (Tellam et al. 1997).

19

Munc18-c was previously implicated as a negative regulator of Syn-4 following Munc18-c

overexpression which impaired GLUT-4 translocation. However, this defect was reversed if

cells were subsequently overexpressed with Syn-4 (Cheatham 2000). Later studies of Munc18-c

(-/+) mice islets revealed impairments in glucose stimulated insulin secretion (Oh et al. 2005)

that were selective to the second phase of release (Oh, Thurmond 2009). These Munc18-c

deficient mice were also more susceptible to type-2 diabetes, supporting a positive secretory

function for this SNARE isoform. Munc18-c appears to closely mediate Syn-4 accessibility to

VAMP-2, which has implications for granule docking at the plasma membrane after glucose

stimulation (Oh, Thurmond 2009).

Munc18-b (Munc18-2) can interact with Syn-1, -2 and -3 and is found abundantly in non-

neuronal cells (Hata, Sudhof 1995). This remaining SM protein isoform has been reported to

regulate trafficking and exocytosis in platelets (Schraw et al. 2003), mast cells (Tadokoro et al.

2007), and epithelial cells (Riento et al. 1998). Most recently, Munc18-b was also implicated in

promoting SNARE complex formation to facilitate vesicle docking and fusion in epithelial

secretion (Liu et al. 2007). In the pancreatic beta cell however, the function of Munc18-b during

insulin release remains to be fully elucidated. Since Munc18-b is upregulated in Munc18-a

knockdown PC12 cells (Arunachalam et al. 2008), Munc18-b may share redundant roles with

Munc18-a in the secretory process. It was also shown that Munc18-b binding to Syn-3 was

functionally important in the transport of vesicles in epithelial cells (Kauppi, Wohlfahrt &

Olkkonen 2002). It is thereby possible that Munc18-b interaction with Syn-3 may serve a

significant role in the exocytotic release of insulin in beta cells as well.

20

1.7 Cellular Functions of Syntaxin-3 in Various Tissues

The Syn-3 SNARE protein is ~33 kDa in size and can exist as isoforms Syn-3A, B, C,

and D following alternative gene splicing. Syn-3A is found exclusively in non-neuronal tissues

while Syn-3B is only expressed in the mouse retina (Curtis et al. 2008). Differential localization

of Syn-3 splice variants may contribute to distinct functions in various cell types. One major

role of Syn-3 is in the regulation of cellular growth and development. Syn-3 proteins are

enriched in cells undergoing rapid growth and differentiation (Martin-Martin et al. 1999, Bajohrs

et al. 2005), and are essential for the outgrowth of neurites (Darios, Davletov 2006). In addition,

Syn-3 has been identified as a target for mediating omega-3 and -6 fatty acid induced cell

membrane expansion (Darios, Davletov 2006). Most recently, omega-3 docosahexaenoice acids

were reported to promote Syn-3 pairing with SNAP-25, which regulates rhodopsin trafficking

and expansion of the ciliary membrane in rod photoreceptor cells (Mazelova et al. 2009).

Numerous studies have demonstrated the versatile role of Syn-3 in regulating the

secretory process of various cell types. In canine kidney cells, Syn-3 has been implicated in

apical membrane docking and fusion events involved in cargo trafficking following a raft based

mechanism (Lafont et al. 1999). In gastric glands, Syn-3 has been shown to regulate parietal

acid secretion in complex with Munc18-b and SNAP-25 (Ammar et al. 2002). In mouse retina

cells, Syn-3 mediates presynaptic transmitter release from conventional and ribbon synapses

(Curtis et al. 2008, Sherry et al. 2006). Interestingly, Syn-3 may also share functional

similarities with Syn-1 as demonstrated in SNARE overexpression studies. Elevated levels of

Syn-3 in insulinoma beta cell lines produced inhibitory effects on exocytosis, Ca2+ channels and

21

insulin biosynthesis that were similar to the results of overexpressed Syn-1 (Kang et al. 2002).

Therefore, the secretory role of Syn-3 may be equally as important as Syn-1, and a study recently

published suggests that Syn-3 functions as a core component of the minimal machinery required

to drive fusion between liposomes in mast cell exocytosis (Sakiyama et al. 2009).

 

1.8 Rationale and Hypothesis – Syntaxin-3 Recruitment of Newcomer Insulin Granules

during Biphasic Glucose Stimulated Insulin Secretion

Previous investigations have revealed a significant role for Syn-3 in the regulation of

exocytosis in pancreatic acinar cells. In acinar cells, zymogen granule exocytosis involves both

primary fusion at the plasma membrane and homotypic fusion within the cytoplasm (Edwardson,

An & Jahn 1997, Hansen, Antonin & Edwardson 1999, Wasle, Edwardson 2002). Syn-3 has

been localized primarily to the zymogen granule membrane, and therefore proposed to function

in secondary granule-granule fusion (Pickett et al. 2007). Since homotypic fusion of zymogen

granules accounts for 70% of total granule fusion events in the acinar cell (Nemoto et al. 2001),

the exocytotic role of Syn-3 is crucial in the secretory process in this exocrine pancreatic tissue.

We hypothesize an equally significant function for Syn-3 in the exocytotic pathway of

the endocrine pancreas, and specifically in islet beta cells. Preliminary data shows an abundance

of Syn-3 in mouse, rat and human beta cells, as well as in the rat INS-1 832/13 cell line. Since

Syn-1A and Syn-4 has been implicated in granule docking and release during first phase GSIS

(Ohara-Imaizumi et al. 2007, Spurlin, Thurmond 2006, Lam et al. 2005), we hypothesize that

22

Syn-3 has a functional role during second phase secretion. The recruitment and priming of

granules to new exocytotic sites on the plasma membrane during second phase GSIS is currently

undefined. However, the increased efficacy of second phase release is in part attributed to

recruitment and accelerated granule-granule fusions that underlie sequential and compound

exocytoses (Kwan, Gaisano 2005). We suggest the molecular machinery for granule-granule

fusions underlying the above exocytotic events involves Syn-3. Since Syn-3 is also the only

exocytotic syntaxin uniquely present on the secretory insulin vesicle (Wheeler et al. 1996), we

hypothesize Syn-3 participates in SNARE mediated fusion between granules.

Employing viral vectors and siRNA strategies, we overexpressed and depleted Syn-3 in

primary beta cells and insulinoma cells models. Using perifusion, secretion assays and imaging

techniques, we examined the effects of Syn-3 perturbation on glucose stimulated insulin

secretion. We also investigated functional differences between Syn-3 in open versus closed

protein conformation. We predict that altered expression of Syn-3 will translate to

corresponding changes in glucose stimulated insulin secretion. We aim to elucidate the roles of

Syn-3 in the primed release of secretory insulin vesicles during alternate exocytotic pathways of

recruitment and granule-granule fusion.

23

Chapter 2

Research Design and Methods

2.1 INS-1 (832/13) Cell Culture

The rat INS-1 (832/13) cell line was donated by Dr. Christopher Newgard (Duke

University Medical Centre, Durham NC). This particular model was chosen for study because

this cell line was characterized by robust KATP channel-dependent and independent glucose

insulin secretion (Hohmeier et al. 2000). INS-1 cells were cultured in RPMI 1640 Medium

(GIBCO) supplemented with 2mM L-glutamine, 10mM HEPES, 10% FBS, 100U/mL penicillin,

100ug/mL streptomycin, 1mM sodium pyruvate and 50µM β-mercaptoethanol at 37ºC in 5%

CO2.

2.2 Immunoblotting

Clonal cell lines or islets were transferred to ice and washed with cold PBS. Cells were

then resuspended with lysis buffer containing protease inhibitors (Roche Applied Science).

Samples were lysed upon sonication and after centrifuging yielded supernatants which were

retained and subjected to 15% SDS-PAGE. Separated proteins were transferred to PVDF

membranes and immunoblotted with anti-Syn1 (Sigma) or anti-Syn3 (SYSY) primary

antibodies, and peroxidase-labeled second antibodies. Protein bands were then visualized by

chemiluminescene (Pierce).

24

2.3 siRNA Transfection of INS-1 Cells

Two days prior to transfection, INS-1 cells were seeded in 12-well plates in RPMI 1640

culture medium supplemented with 2mM L-glutamine, 10mM HEPES, 1mM sodium pyruvate

and 50µM β-mercaptoethanol at 37ºC in 5% CO2. On the day of transfection, 0.2mL of OPTI-

MEM® I medium (Invitrogen) was mixed with 0.6µL of RNAi duplex (Dharmacon) solution

(20µM) in a microfuge tube. The siRNA pool targeting the rat Syntaxin-3 gene contained the

following nucleotide sequences:

5’-GAGAUUGAGGGUCGGCACA-3’, 5’-AAACGAGGCUCAACAUCGA-3’,

5’-CUGAAAUAAGAGUGGCCUA-3’ and 5’-GAAUCAGGGUGAGAUGUUA-3’.

The non-targeting pool (NTP) of siRNAs used in control cells contained the following nucleotide

sequences: 5’-UGGUUUACAUGUCGACUAA-‘3, 5’-UGGUUUACAUGUUGUGUGA-‘3,

5’-UGGUUUACAUGUUUUCUGA-‘3 and 5’-UGGUUUACAUGUUUUCCUA-‘3.

In another microfuge tube, 50µL of OPTI-MEM® I medium was combined with 2µL of

Lipofectamine® RNAiMAX (Invitrogen). The contents of the two microfuge tubes were mixed

together and incubated at room temperature for 10-20min. Approximately 0.84mL of this

mixture was then added to each well. Cells were then incubated for 48 hours before being

collected for static secretion assay or immunoblotting.

2.4 Static Insulin Secretion Assay

Adenoviral transduced INS-1 cells grown in 12-well plates in RPMI 1640 culture medium

were washed once with Krebs-Ringer bicarbonate (KRB) buffer and incubated in 1mL of KRB

solution for one hour. Buffer was aspirated and cells were incubated in 1mL of 0.8mM glucose

KRB solution for one hour. Fractions were then collected and 1mL of 10mM glucose KRB

25

solution was added to each well. After incubating for one hour, fractions were collected. INS-1

cells were then trypsinized and pelleted by centrifugation. Fractions of secreted insulin and

lysed cell samples were then quantified for insulin using a radioimmunoassay (RIA) kit (Linco

Research Inc.) according to manufacturer’s instructions.

2.5 Lentivirus Transduction and Establishment of New Stable Cell Lines

All lentiviruses used in this study were prepared by Dr. Shuzo Sugita’s laboratory

(University of Toronto). Two days before transduction, INS-1 cells were plated in 100mm

dishes and incubated in RPMI 1640 growth medium at 37ºC in 5% CO2. On the day of

transduction, INS-1 cells were transduced with equal volumes of lentivirus solution and RPMI

1640 medium. Cells were then incubated for 48 hours at 37ºC in 5% CO2. After 2 days,

medium containing lentiviruses was discarded and replaced with fresh culture medium. Cells

were then incubated in virus free medium for 48 hours at 37ºC in 5% CO2. Puromycin selection

was then used to isolate successfully transduced cells by adding 10mL of 1µg/mL puromycin

RPMI 1640 medium to each dish. Cells were then passaged and maintained in growth medium

containing puromycin for ~2 weeks in order to yield a stable cell line.

2.6 Total Internal Refraction Fluorescence Microscopy

TIRFM was performed by Dr. Dan Zhu from Dr. Herbert Gaisano’s laboratory (University

of Toronto). TIRF imaging uses an evanescent wave, which results from the reflection of

incident light at the boundary between two mediums of different refractive indexes (Figure 5).

Since the evanescent wave field only penetrates a depth of ~100nm from the cell plasma

membrane, TIRFM allows for the selective visualization of this region in high resolution. INS-1

26

cells were cultured in RPMI 1640 supplemented with 10% FBS at 37oC and 5% CO2. One day

after plating, cells were transfected with Syntaxin3-RNAi or NTP control using Lipofectamine®

RNAiMAX (Invitrogen). One day after transfection, INS-1 cells were infected with either

Syncollin-pHluorin or Adenovirus-NPY-EGFP. Syncollin-pHluorin consists of granule

membrane anchored protein, Syncollin, fused to a pH-sensitive form of EGFP in the vesicle

lumen. In the acidic environment of the granule lumen, Syncollin-pHluorin is non-fluorescent.

However, upon fusion of insulin vesicles with the plasma membrane during exocytosis, exposure

to the neutral extracellular environment causes EGFP to fluoresce. Therefore, each bright spot

on the cell represents the exocytosis of a secretory granule. By measuring the number and

fluorescence intensity of these bright spots on the plasma membrane, changes in exocytosis can

be elucidated (Figure 6).

Ad-NPY-EGFP, consisting of EGFP tagged Neuropeptide-Y, is a vesicle cargo protein

which was also used to visualize insulin granules located near the plasma membrane. Since

NPY-EGFP is not pH-sensitive, this fluorescent marker allowed for the differentiation between

the fusion of pre-docked vs. newcomer granules. Experiments were performed 48 h after

transfection at room temperature in a standard extracellular saline solution containing 138 mM

NaCl, 5.6mM KCl, 1.2mM CaCl2, 2.6mM MgCl2, 5mM glucose and 5 mM HEPES (pH 7.4). A

16.7mM high glucose solution used to stimulate the INS-1 cells was enhanced with 10nM GLP-1

and 150μM isobutylmethylxanthine (IBMX).

In the TIRF microscopy setup, a 488-nm beam was used to excite EGFP or pHluorin,

combined with a 488RDC long-pass dichroic filter and a 525/50-nm band-pass emission filter.

The red fluorescence was excited by a 543-nm beam combined with a 543-nm RDC dichroic

filter and a 600/75-nm band-pass emission filter cube (Chroma). Dual images of green and red

27

fluorescence were split by the Dual-View image splitter and recorded using the cooled 16-

bit EM-CCD camera (Cascade Quant-EM) at the right port of the microscope. The penetration

depth of the evanescent field (130 nm) was aligned by measuring the incidence angle of the 488-

nm laser beam with a prism (n = 1.5163). Images were acquired at 5-Hz with a 100-ms exposure

time by a Nikon NIS-Elements software. Insulin granule mobilities and exocytosis were

analyzed by Matlab (MathWorks), ImageJ (NIH), Igor Pro 5.01 (WaveMetrics) softwares.

28

Figure 5. Schematic drawing of the evanescent field by TIRFM. An evanescent wave arises on the cell-substrate interface and penetrates a very small distance into the cell, allowing for the behaviour of insulin granules near the plasma membrane to be tracked with precision. Note: n2 must have a lower refractive index than n1. (Figure from unknown source)

29

Figure 6. TIRFM of insulin granules labelled with Syncollin-pHluorin. Top: The Syncollin-pHluorin signal footprint of a cell undergoing exocytosis after K+-stimulation. Bottom: The corresponding fluorescence intensity change of the whole cell (background subtracted) shown. The grey lines indicate the images acquired at the corresponding time points.

30

2.7 Cell Preparation for Confocal Immunofluorescence Microscopy

One day before preparation for imaging, cells were seeded onto coverslips and incubated

overnight at 37ºC in 5% CO2. The following day, cells were washed with PBS and fixed with

3.7-4% paraformaldehyde for 15 min at room temperature. After fixing, cells were

permeabilized with 0.1% Triton X100 for 15 min at room temperature. Cells were then blocked

for non specific proteins with 10% goat serum solution for 1 hour at room temperature. After

washing with PBS, cells were immunostained with primary antibodies for 1-2 hours, followed by

secondary antibody incubation for 0.5-1 hour. Coverslips were then mounted onto microscope

slides with fluorescent mounting medium (DAKO).

2.8 Adenovirus Production and Purification

The EGFP, Syn-1A and Syn-3 adenoviruses used in this study were prepared by Yu He

from Dr. Herbert Gaisano’s laboratory (University of Toronto). The empty virus control was

donated by W. Hughes (Garvan Institute, Sydney, Australia). Recombinant Syn-1A and Syn-3

adenoviruses were prepared using the Gateway® pAD system (Invitrogen) and subjected to

plaque-purification and amplification. Constitutively open form mutants of Syn-1A and Syn-3

were generated through a double mutation in Leu-165 Ala and Glu-166 Ala in the wild

type protein (Dulubova et al. 1999) (Figure 7).

31

Syn-1A 1 MKDRTQELRTAKD-S-DDD-DDVTVTVDRDRFMDEFFEQVEEIRGFIDKIAENVEEVKRK Syn-3 1 MKDRLEQLK-AKQLTQDDDTDEVEIAIDNTAFMDEFFSEIEETRLNIDKISEHVEEAKKL **** * ** *** * * * ****** ** * **** * *** * Syn-1A 58 HSAILASPNPDEKTKEELEELMSDIKKTANKVRSKLKSIEQSIEQEEGLNRSSADLRIRK Syn-3 60 YSIILSAPIPEPKTKDDLEQLTTEIKKRANNVRNKLKSMEKHIEEDE-V-RSSADLRIRK * ** * * *** ** * *** ** ** **** * ** * ********** Syn-1A 118 TQHSTLSRKFVEVMSEYNATQSDYRERCKGRIQRQLEITGRTTTSEELEDMLESGNPAIF Syn-3 118 SQHSVLSRKFVEVMTKYNEAQVDFRERSKGRIQRQLEITGKKTTDEELEEMLESGNPAIF *** ********* ** * * *** ************ ** **** ********** Syn-1A 178 ASGIIMDSSISKQALSEIETRHSEIIKLENSIRELHDMFMDMAMLVESQGEMIDRIEYNV Syn-3 178 TSGII-DSQISKQALSEIEGRHKDIVRLESSIKELHDMFMDIAMLVENQGEMLDNIELNV **** ** ********** ** * ** ** ******** ***** **** * ** ** Syn-1A 238 EHAVDYVERAVSDTKKAVKYQSKARRKKIMIIICCVIL-GIIIASTIG Syn-3 237 MHTVDHVEKARDETKRAMKYQGQARKKLIIIIVIVVVLLGIL-ALIIG * ** ** * ** * *** ** * * ** * * ** * **

Figure 7. Rat Syntaxin-1A and Syntaxin-3 protein sequence alignment. Alignment of the amino acid sequences from rat (Rattus norvegicus) Syn-1A (M95734) and Syn-3 (L20820). Optimal alignment was produced with the ExPASy Proteomics SIM program. Conservation between Syn-1A and Syn-3 is indicated by the residues marked with asterisks. Highlighted residues indicate the mutation sites of wild-type Syntaxin to generate the open-form mutant. In both Syn-1A and Syn-3 open form mutants, Leu-165

Ala and Glu-166 Ala.

32

2.9 Adenoviral Transduction of INS-1 Cells

INS-1 cells were grown in 12-well plates in RPMI 1640 culture medium one day prior to

transduction. The next day, old medium was removed and replaced with 0.3mL of fresh medium

containing adenoviruses and incubated at 37ºC in 5% CO2 for 1.5 hour. Medium was then

discarded and replaced with fresh virus free RPMI 1640 medium. Cells were then incubated at

37ºC in 5% CO2 for 48 hours and subject to static insulin secretion assay, immunoblotting or

confocal microscopy.

2.10 Isolation of Mouse Pancreatic Islets and Beta Cells

Male C57BL/6 mice, 19-21 in bodyweight (Charles River Laboratories Inc.), were

sacrificed by cervical dislocation and the abdominal cavity was opened. Under a dissecting

microscope, the pancreatic duct was cannulated and 2mL of 2mg/mL collagenase (Sigma) was

injected into the common bile duct for pancreatic digestion. The pancreas was then excised and

incubated at 37ºC for 17min, followed by shaking to break apart tissue. The pancreatic samples

were then centrifuged at 1400rpm for 2 min at 4ºC, washed with Hanks’ balanced salt

solution/HEPES and filtered through a gauze. The islets were then handpicked under an upright

stereomicroscope and cultured in RPMI 1640 medium supplemented with 11mM D-glucose,

10% FBS, 100U/mL penicillin and 100µg/mL streptomycin at 37ºC in 5% CO2 overnight.

To further isolate single beta cells, islets were washed with PBS and centrifuged at

1000rpm for 4 min at room temperature. Islets were then trypsinized and incubated for 5 min at

37ºC in 5% CO2. Using a pipette, islets were then dispersed into individual cells and incubated

in 2.8mM glucose RPMI 1640 containing FBS at 37ºC in 5% CO2.

33

2.11 Adenoviral Transduction of Mouse Islets

Mouse islets were collected and placed in 35mm dishes containing 1mL of 11mM glucose

RPMI 1640 medium. Adenoviruses were then pipetted into dishes and incubated at 37ºC in 5%

CO2 for 1.5 hours. Medium was then removed and replaced with fresh 11mM glucose RPMI

1640 and incubated for 48 hours at 37ºC in 5% CO2 before perifusion or immunoblotting.

2.12 Mouse Islet Perifusion

Groups of 50 islets were placed in perifusion chambers and perifused at 37ºC with Krebs-

Ringer bicarbonate buffer (pH 7.4) supplemented with 10mM HEPES and 0.07% bovine serum

albumin at a flow rate of ~1mL/min. Islets were equilibrated for 30min in KRB buffer

containing 2.8mM glucose, followed by 10min of 2.8mM glucose stimulation and 40min of

16.7mM glucose stimulation. Eluted fractions were collected and quantified for insulin content

using a RIA kit (Linco Research Inc.) following manufacturer’s instructions. Islets were

retrieved following perifusion and pelleted by centrifugation and lysed using acid-ethanol

solution (0.2mM HCl in 75% ethanol). Islet lysates were quantified for total insulin content and

used to normalize secreted insulin values.

2.13 Statistics

All data are presented as the mean ± standard error of the mean (SEM) with the indicated

number of experiments. Statistical significance was evaluated by the Student’s t-test using

Microcal Origin 6.0. In TIRFM analysis, Mann-Whitney rank sum test was performed according

to the normality of datum distribution in SigmaStat 3.11 (Systat Software, Inc). A significant

difference is indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001).

34

Chapter 3

Results 3.1 Syntaxin-3 Expression in Pancreatic Islets and Colocalization with Insulin Granules

We first performed immunoblot analysis to confirm the abundant levels of Syn-3 in

mouse pancreatic islets as shown in Figure 8A. Next, we examined the subcellular localization

of endogenous Syn-3 in beta cells using confocal microscopy. After dispersion of islets into

single cells, one group of beta cells remained in regular culture medium, while a second group

was stimulated with glucose and GLP-1 for three minutes. Cells were then immediately fixed

and immunostained with antibodies against Syn-3 and insulin. Analysis of unstimulated beta cell

images showed Syn-3 to be more or less evenly dispersed and colocalized to insulin granules

near the plasma membrane (Figure 8C, top row). In stimulated conditions, Syn-3 was present in

higher concentration at the plasma membrane and this was seen as a more clearly defined cell

shape. Stimulated beta cells also had larger Syn-3 clusters in the cytosol and plasma membrane

area, many of which were colocalized to insulin granules (Figure 8C, bottom row). The Pearson

correlation coefficient for Syn-3 and insulin granules was calculated to be 0.79 ± 0.011 (n=19

cells) indicating a strong localization to insulin granules (Figure 8B). These clusters likely

correspond to the homotypic fusion of insulin granules that is characteristic of GLP-1 stimulated

secretion (Kwan, Gaisano 2005) and electron microscopy ultrastructural studies will be needed

to confirm this. Furthermore, the concentrated levels of Syn-3 at the plasma membrane

following stimulation also support the notion of essential Syn-3 trafficking to the target

membrane in order to facilitate insulin release.

35

A)                                                              B)   

 

 

 

C) 

Figure 8. Endogenous expression of Syntaxin-3 in mouse pancreatic islets and beta cells. (A) 2µg of rat brain lysate (as a positive control) and (20µg of protein) from pancreatic mouse islets were prepared for use in anti-Syn3 immunoblotting to examine endogenous Syn-3 expression. (B) Distribution of calculated Pearson coefficient values between Syn-3 and insulin granules in mouse beta cells (n=19). (C) Representative images of mouse beta cells captured with confocal immunofluorescence microscopy in unstimulated conditions (top row) and during glucose and GLP-1 stimulated secretion (bottom row). The left column displays images stained for Syn-3, the middle column displays images stained for insulin and the right column displays the merged images.

36

3.2 RNAi-mediated Depletion of Syntaxin-3 Impaired Glucose Stimulated Insulin Secretion

in INS-1 Cells

To examine the endogenous function of Syn-3 in secretion by INS-1 cells, we used RNA

interference (RNAi) to downregulate Syn-3 expression through post transcriptional gene

silencing. After entry into the cell, the RNA interference pathway begins when double stranded

RNA is cleaved by the Dicer enzyme into smaller fragments 21-23 nucleotides long (Ketting et

al. 2001). These small interfering RNA (siRNA) strands are incorporated into the RNA-induced

silencing complex (RISC) and interact with a complementary region of messenger RNA. RISC

cleaves the bound mRNA which then promotes the degradation of this mRNA segment by

exonucleases. As a result, target gene expression is effectively repressed (Dykxhoorn, Novina &

Sharp 2003). In our study, the introduction of siRNA against Syn-3 depleted protein levels by

~70% and did not affect Syn-2, VAMP-2 or VAMP-8 expression (Figure 9A i). Following

glucose stimulation in a static secretion assay, diminished Syn-3 expression evoked a ~50%

reduction in insulin release, compared to control cells treated with non-targeting siRNAs (Figure

9B). Syn-3 RNA silencing significantly impaired glucose stimulated insulin release (ratio of

GSIS to insulin content: 0.087 ± 0.003% for si-Con vs. 0.051 ± 0.004% for si-Syn3, p<0.01), but

had no effect on basal secretion (0.39 ± .022% for si-Con vs. 0.57 ± 0.22% for si-Syn3) or

cellular insulin content (1.12 ± 0.04% for si-Con vs. 1.10 ± 0.05% for si-Syn3, relative to

untransfected control). Taken together, these data show an important role for Syn-3 in

facilitating glucose stimulated insulin secretion.

37

B)                    Figure 9. RNAi-mediated depletion of Syntaxin-3 impaired glucose stimulated insulin secretion. (A) i) INS-1 cells were transduced with si-Con or si-Syn3 and prepared for use in anti-Syn3 immunoblotting to detect knockdown efficiency. INS-1 cell lysates were also prepared for use in immunoblotting to examine for changes in Syn-2, VAMP-2 and VAMP-8 protein expression. ii) Optical density scanning quantification was used to derive the band density for each si-Con band, which was used to normalize the si-Syn3 bands in each experiment following immunostaining for Syn-3. Data represent the average ±SE of three independent experiments; **p<0.01 (B) 24h following adenovirus infection, cells were pre-incubated in KRBH for one hour, followed by an hour of stimulation in 0.8mM and 10mM glucose conditions. Secreted insulin was measured by RIA. Data represent the average ±SE from three independent experiments; **p<0.01

 

38

3.3 Diminished Glucose Stimulated Insulin Secretion in Stable Syntaxin-3 Knockdown

Cells

We then established a stable Syn-3 knockdown cell line to investigate the effects of

persistent Syn-3 gene silencing. To repress Syn-3 expression, we used short hairpin RNA

(shRNA) to once again trigger the RNAi pathway. In this case, shRNA was favoured over

siRNA due to its characteristic stable expression in cells (Paddison et al. 2004). The pLKO-puro

vector system was used to stably express shRNA in INS-1, and select for infected cells with

puromycin antibiotic. pLKO is a HIV-1 based expression vector which contains the required

viral processing elements to effectively integrate into the host genome (Zufferey et al. 1997).

After incorporation into the nuclear DNA, the expression of shRNA is driven by the human U6

promoter (Paddison et al. 2004). Lentiviruses encoding a puromycin resistance gene and either

Syn-3 shRNA or EGFP, were used to infect INS-1 cells. This allowed us to generate Syn-3

knockdown clones and a control cell line. After fluorescent verification of transduction

efficiency (Figure 10A), puromycin resistant cells were selected for over a one month period.

Immunoblot analyses of remaining cells revealed knockdown to be heterogeneous as Syn-3

expression was reduced by ~50% (Figure 10B). This culminated to a significant (p<0.01) 40%

decrease in GSIS during a static insulin secretion assay comparing the glucose response between

Syn-3 KD and control groups (Figure 11). To examine whether Syn-3 depletion altered the

expression of other syntaxin isoforms and exocytotic proteins, Western blotting was performed

on the cell lysates of knockdown clones. Compared to the control cell line, we found decreased

expression of Syn-2 by ~34% and VAMP-2 by ~46% in Syn-3 KD cells, while the levels of

other exocytotic proteins remained unchanged (Figure 12).

39

 A)   

     B) INS‐1 lysate 

  Figure 10. Stable Syntaxin-3 knockdown INS-1 clones established using lenti-shRNA. (A) Representative brightfield image and corresponding fluorescent signal from INS-1 cells after infection with lenti-pLKO-EGFP and puromycin selection of successfully transduced cells. (B) Lysates of INS-1 cells stably expressing EGFP (Control) and Syn-3 shRNA (Syn-3 KD) were prepared for use in anti-Syn3 immunoblotting to detect knockdown efficiency. Optical density scanning quantification was used to derive the band density for each Control band, which was used to normalize the Syn3 KD bands in each experiment. Data represent the average ±SE of three independent experiments; **p<0.01    

40

                      Figure 11. Diminished glucose stimulated insulin secretion in stable Syntaxin-3 knockdown cells. The stable cell lines were pre-incubated in KRBH for one hour, followed by an hour of stimulation in 0.8mM and 10mM glucose conditions. Secreted insulin was measured by RIA. Data represent the average ±SE from three independent experiments; **p<0.01

         

41

A)  

                  B) Syn‐2  

  

 

    

 

       C) VAMP‐2 

 

  

Figure 12. Immunoblot analyses examining exocytotic protein expression. (A) Cell lysates containing 20µg of protein from control and Syn-3 KD INS-1 cells were immunostained for various exocytotic proteins, and changes in expression level between control and KD cells were compared. Samples of rat brain (2µg) and acinar cells (10µg) were used as positive and negative controls respectively. (B, C) Optical density scanning quantification was used to derive the band density for each Control band, which was used to normalize the Syn-3 KD bands following immunostaining for Syn-2 in (B) and VAMP-2 in (C). Data represent the average ±SE of three independent experiments; *p<0.05, **p<0.01   

42

3.4 Syntaxin-3 Depletion Reduced Newcomer Granule Mobilization Underlying Biphasic Insulin Secretion To elucidate whether changes in granule dynamics contributed to the secretion deficits

seen in Syn-3 knockdown cells, total internal reflection fluorescence microscopy (TIRFM) was

performed on siRNA treated INS-1 groups. After labelling insulin vesicles with Syncollin-

pHluorin, TIRFM allowed us to selectively visualize the fusion of fluorescent granules at the

plasma membrane. Our results showed the accumulated fusion events in Syn-3 silenced cells

were significantly reduced during latter half of first phase release, and for the duration of second

phase secretion (Figure 13B). Compared to control, the average fusion rate of Syn-3 silenced

cells in both the first and second phase of secretion was decreased by ~40-45% (first phase: 15.6

± 2.42 per cell per 100 µm2 for control vs. 9.97 ± 1.27 per cell per 100 µm2 for Syn3-silenced

cells, p<0.05; second phase: 8.36 ± 1.38 per cell per 100 µm2 for control, and 4.09 ± 1.00 per cell

per 100 µm2 for Syn3-silenced cells, p<0.03) (Figure 13C). Lowered fusion rates in TIRFM

correspond to fewer fusion events at the plasma membrane. However, to elucidate whether

decreased secretion pertained to impaired fusion of pre-docked or newcomer granules,

experiments were repeated with insulin vesicle cargo marker, NPY-EGFP.

Insulin granules containing NPY-EGFP were observed as bright spots on the cell

membrane as shown in Figure 14. By measuring the intensity and duration of the fluorescent

signal emitted from these vesicles, we were able to differentiate between pre-docked vs.

newcomer granule fusion modes (Figure 14 i,ii,iii). After infecting INS-1 with Ad-NPY-EGFP

but prior to stimulation, we calculated the vesicle density of granules on the plasma membrane

and found no differences between control and Syn-3 depleted cells (0.105 ± 0.0058 vesicles per

µm2 for control vs. 0.103 ± 0.00678 vesicles per µm2 for Syn-3 KD) (Figure 15). Upon glucose

43

stimulation, the exocytosis of pre-docked granules was also comparable between both groups as

shown in Figure 16C (blue bars). This indicated that the number and fusion competency of

docked and primed vesicles at the plasma membrane was preserved, notwithstanding Syn-3

depletion. However, the exocytosis of newcomer granules, including those that were docked

briefly (green bars in Figure 16C) or not docked at all (red bars in Figure 16C), was significantly

decreased during both phases of secretion in Syn-3 KD cells. This reduction likely corresponds

to the decelerated recruitment of insulin granules to refill the RRP. The results of accumulated

fusion events in control vs. Syn-3 KD cells was consistent with earlier data obtained from

experiments with Syncollin-pHluorin, in which Syn-3 depletion effectively impaired biphasic

secretion (first phase: 13.0 ± 1.79 fusion events per 100 µm2 for control vs. 5.4 ± 1.01 fusion

events per 100 µm2 for Syn-3 KD, p<0.01; second phase: 11.1 ± 3.38 fusion events per 100 µm2

for control, and 4.53 ± 1.31 fusion events per 100 µm2 for Syn-3 KD, p<0.05) (Figure 16A,B).

Taken as a whole, our data suggests that Syn-3 depletion evoked a reduction in newcomer

granules and their subsequent exocytotic fusion, thereby impairing biphasic insulin secretion.

 

44

Figure 13. Syntaxin-3 depletion reduced the total sum of fusion events underlying biphasic insulin secretion. (A) Histogram of the number of fusion events (per 100 μm2) in control INS-1 cells (top; 10 cells) and Syn3-siRNA-silenced INS-1 cells (bottom; 10 cells) at 30s intervals. Time 0 indicates when 22 mM high glucose plus 10 nM GLP-1 and IBMX were added. (B) Time-lapse curves of accumulative fusion events per cell per μm2 display the difference between the control group and Syn3-siRNA-silenced group. (C) Comparison of the number of fusion events (per cell per 100 μm2) at 1st phase (0-5 minutes after stimulation) or 2nd phase (5-18 minutes), respectively; *p<0.05 for 1st phase, *p<0.03 for 2nd phase.

45

Figure 14. TIRFM of INS-1 cells expressing NPY-EGFP insulin vesicle cargo protein differentiated between pre-docked and newcomer granule fusion modes. Representative images of a control and Syn-3 KD INS-1 cell by TIRFM after 2-day co-transfection with Syn-3 siRNA and NPY-EGFP. Scale bar: 2mm. The sequential images and corresponding time-lapse fluorescence intensity curves indicate different fusion modes: (i) A fusion event from a pre-docked insulin granule. (ii) A fusion event of a newcomer granule which did not undergo a docking step on the plasma membrane before proceeding to exocytosis. (iii) A fusion event of a newcomer granule that underwent a short docking time (32 s in this case) on the plasma membrane before exocytosis.

46

Figure 15. Syntaxin-3 depletion did not affect the vesicle density at the plasma membrane prior to stimulation. TIRFM of control and Syn-3 KD INS-1 cells 2 days after infection with Ad-NPY-EGFP showed no significant differences in the mean granule densities at the plasma membrane prior to stimulation (n= 20 cells).

47

Figure 16. Syntaxin-3 depletion reduced newcomer granule mobilization underlying biphasic insulin secretion. Control and Syn-3 knockdown (KD) INS-1 cells were stimulated with 16.7 mM glucose plus 10 nM GLP-1 plus 150 mM IBMX and analyzed by TIRFM. (A) The normalized cumulative fusion events of insulin granules per cell from control cells and Syn-3 KD INS-1 cells. (B) Summary of fusion events from A) in the first phase (0 - 5 min) and second phase (5 - 20 min) secretion. ***p<0.001 for 1st phase, *p<0.05 for 2nd phase. (C) Analysis of docked vs. newcomer granule exocytosis during biphasic insulin secretion from control cells (left) and Syn-3 KD (right) INS-1 cells. Note: pre-docked granules (blue) exocytosis is similar between control and Syn-3 KD cells; no-dock (red) and short-dock (green) newcomer granules are reduced in Syn-3 KD in both first and second phases. (n=10 cells, 338 fusion events from control group; n=12 cells, 206 fusion events from Syn-3 KD group)

48

3.5 Syntaxin-3 Overexpression Potentiated Glucose Stimulated Insulin Secretion in Mouse

Islets

Our next aim was to determine how elevated Syn-3 levels would affect the insulin

secretion of glucose perifused mouse islets. However, before initiating the perifusion assay,

numerous measures were taken to test the transduction efficiency of our adenoviruses. First,

Western blotting with anti-Syn3 and anti-EGFP antibodies was performed to verify the

successful expression of viral delivered proteins in mouse islets (Figure 17A). Next, infected

islets were dispersed into single cells and the number of fluorescent cells were compared to the

total cell count, yielding a transduction efficiency of ~30% (Figure 17B,C,D). Lastly, confocal

imaging was used to generate z-slices through whole islets to show fluorescence in the islet

centre, confirming viral penetration to the islet core where the majority of beta cells are situated

(Brissova et al. 2005) (Figure 18).

Perifusion analyses of Ad-Syn3 transduced islets showed a dramatic increase in the first

and second phase of stimulated secretion, while constitutive basal insulin release remained

unchanged (Figure 19A). The characteristic biphasic secretion curve of Ad-Syn3 was amplified

in the peak and plateau area, representing enhanced secretion in the transient first phase and

sustained second phase of insulin release. Comparison of area under the curve during first phase

(6.57E-3 ± 7.51E-4 for Ad-Syn3 vs. 3.09E-3 ± 4.22E-4 for control, p<0.05, n=3-6) and second

phase (4.67E-3 ± 6.80E-4 for Ad-Syn3 vs. 2.15E-3 ± 1.22E-4 for control, p<0.05, n=3-6) further

revealed the marked potentiation of >75% in biphasic insulin release (Figure 19B). To examine

whether enhanced secretion was secondary to higher insulin content, islet groups were measured

49

for total insulin using RIA. Measured readings indicated no differences in cellular insulin

content between Ad-Syn3 transduced islets and the control group (25.14 ± 1.08 ng/islet for Ad-

Syn3 vs. 27.96 ± 3.80 ng/islet for control, n=3) (Figure 20). These results are consistent with our

data from knockdown studies, providing further evidence for the positive role of Syn-3 in

regulating biphasic insulin secretion.

   

50

A) 

  

   Figure 17.  Syntaxin-3 overexpression and transduction efficiency in mouse islets. (A) One hour after isolation, mouse islets were infected with Ad-EGFP (control), Ad-EGFP-Syn3-WT. After 24h incubation, cell lysates were immunoblotted with anti-EGFP and anti-Syn3 to confirm the expression of viral delivered proteins at 62kDa and 27kDa and endogenous syntaxin levels at 35kDa. (B) Islets were dispersed into single cells 24h after infection and the number of fluorescent cells were counted under a microscope and compared to the total number of dispersed cells in order to determine transfection efficiency. Data represent the average ±SE of three independent experiments. (C) Representative image of whole islets infected with Ad-EGFP-Syn3 after an incubation period of 24h. (D) Representative image of single cells that were dispersed from islets 24h after infection.    

51

 

A)              

B) 

   Figure 18. Confocal microscopy of whole mouse islet core. (A) Representative image and (B) z-stack of EGFP signal detected in a mouse islet following adenoviral infection.

  

52

A)           B)          

  Figure 19. Syntaxin-3 overexpression potentiates glucose stimulated insulin secretion in mouse islets. (A) Islets were isolated and handpicked into groups of 50 for adenovirus infection 1h after isolation. After 24h, islets were incubated for 8 minutes at low glucose (2.8mM), followed by basal sample collection (1-10min) at low glucose to establish a baseline. Glucose was then elevated to 16.7mM. Eluted fractions were collected at 1 min intervals and insulin secretion determined by RIA. Insulin concentration values were normalized to the total insulin content of 50 islets perifused in each group. Data shown are the average ±SE from five independent experiments. (B) The average ±SE of area under the curves (AUC) of normalized insulin release from perifusions in (A). Data represent insulin release from 11-26 min for first-phase secretion (FP-IS), 27-40 min for second-phase secretion (SP-IS) and 11-40min minus basal secretion (0-10 min) for total GSIS; *p<0.05

53

           

 

 

  Figure 20. Cellular insulin content of mouse islets. Islets were isolated and handpicked into groups of 50 for adenovirus infection one hour after isolation. After 24h, total insulin content was measured by RIA. Data shown are the average ±SE from three independent experiments.

54

3.6 Overexpression of Syntaxin-3 (in Wild Type Conformation) Enhanced Glucose

Stimulated Insulin Secretion in INS-1 Cells

Since, the open conformation of Syn-1A was previously reported to enhance its

exocytotic actions by promoting SNARE complex formation (Sutton et al. 1998), we constructed

a Syn-3 open form mutant to examine for similar effects on insulin secretion. INS-1 cells were

infected with adenoviruses carrying EGFP tagged syntaxins, and the transduction efficiency was

verified using epifluorescent microscopy (Figure 21A). Successful Syn-3 open form (OF) and

wild type (WT) overexpression was then confirmed with Western blotting (Figure 21B), before

subjecting transduced INS-1 cells to a static secretion assay. Additional controls groups were

established with Ad-EGFP (negative control), and EGFP tagged Ad-Syn1A-OF and Ad-Syn1A-

WT (positive controls).

Following stimulation with glucose, we found that secretion by Ad-Syn-1A cells was

consistent with previous reports in which OF Syn-1A enhanced insulin release (by 63%), while

the wild-type protein had no effect. In contrast, cells overexpressing Syn-3 WT exhibited a 47%

increase in secretion (0.10 ± 0.002 for Syn3 WT vs. 0.07 ± 0.01 for control, normalized to total

insulin content, p<0.05, n=3), while potentiation by exogenous Syn-3 OF was statistically

insignificant. Basal secretion between all groups remained unchanged (Figure 21C). These

results demonstrate how syntaxin conformation may produce differential effects specific to each

isoform. Furthermore, although elevated levels of Syn-3 had enhanced insulin release, our data

revealed the sensitivity of GSIS as exogenous open-form Syn-3 was unable to potentiate

secretion to the same extent as wild-type Syn-3.

55

 

  

 Figure 21. Overexpression of Syntaxin-3 (wild type) enhanced glucose stimulated insulin secretion in INS-1 cells (A) INS-1 cells were infected with Ad-EGFP (control), and wild-type and open form Ad-EGFP-Syn3 and Ad-EGFP-Syn1. After 24h incubation, cell lysates were immunostained with anti-Syn3 and anti-Syn1 to detect the overexpression of viral delivered proteins at 62kDa. (B) Representative image of epifluorescent signal detected in INS-1 cells treated with Ad-EGFP 24h after infection. (C) After 24h incubation following infection by adenoviruses, INS-1 cells were preincubated with 0.8mM glucose followed by stimulation with 10mM glucose. Secreted insulin was measured by RIA. Data represent the average ±SE of three independent experiments with n = 4 for each group; *p<0.05

56

3.7 Mislocalization of Open-Form Syntaxin-3 Following Overexpression in INS-1 Cells

We hypothesized that perhaps exogenous open form Syn-3 had no effect on secretion due

to mislocalization, and employed confocal immunofluorescence microscopy to test our theory.

After infecting INS-1 cells with EGFP tagged adenoviruses used in our overexpression studies,

we stained with anti-insulin antibodies to allow visualization of the secretory granules. Confocal

imaging results showed an abundance of wild type Syn-3 in punctuate pattern along the plasma

membrane that was mostly colocalized with insulin granules. In contrast, open form Syn-3 was

less prevalent on the plasma membrane and more densely clustered in the cytosol and perinuclear

region of the cell. The degree of colocalization between insulin granules and open form Syn-3

mutants was also lower compared to wild-type Syn-3 (Figure 22A). Additional images of

overexpressed Syn-1A were captured to show the high concentration of Syn-1A clearly

delineating the plasma membrane (Figure 22B), which can be contrasted to the more punctuated

plasma membrane clusters of Syn-3. Open form Syn-1A, unlike open form Syn-3, was correctly

targeted to the plasma membrane, although some excess open form Syn-1A became localized to

the insulin granules more so than overexpressed Syn-1A WT. Taken together, our data supports

our hypothesis in which mislocalized Syn-3 OF prevented the mutant proteins from acting on

target membranes to effectively enhance insulin secretion following exogenous overexpression

in INS-1 cells. Nonetheless, the correctly localized overexpressed Syn-3 WT to insulin granules

was able to potentiate Syn-3 actions on exocytosis.

57

A)              

B)                       

 

Figure 22. Mislocalization of open-form Syntaxin-3 following overexpression in INS-1 cells. Representative images of INS-1 cells captured with confocal immunofluorescence microscopy following infection with Ad-Syn-3WT and OF and Ad-Syn-1WT and OF. The left column displays images stained for Syn-3 in (A) and Syn-1 in (B). The middle column displays images stained for insulin and the right column displays the merged images.

58

Chapter 4

Discussion

4.1 Syntaxin-3 Facilitates Exocytosis of Newcomer Granules during Biphasic Glucose

Stimulated Insulin Secretion

In our investigation, overexpression of Syn-3 potentiated GSIS in INS-1 cells while

reduced endogenous Syn-3 impaired secretion, demonstrating a novel role for Syn-3 in positively

regulating glucose stimulated insulin secretion. We found that increasing wild-type Syn-3

enhanced GSIS to a level comparable to that achieved by Syn-1A overexpression. These results

are consistent with our previous report in which Syn-3 mimicked the actions of Syn-1A on L-

type calcium channels, insulin biosynthesis and exocytosis in HIT cells (Kang et al. 2002).

These parallel effects may be due to analogous structure function properties shared between Syn-

3 and Syn1A, such as conserved coiled coil domains and a 64% amino acid homology (Bennett

et al. 1993). In addition, both Syn-3 and Syn-1A can bind to SNAP-25 and VAMP-2 to form a

trimeric SNARE complex (Calakos et al. 1994, Fasshauer et al. 1999), likely contributing to their

shared role in facilitating GSIS. The mechanisms by which each isoform enhances secretion,

however, may differ considerably, as further investigation with perifusion and TIRFM revealed

separate, non-redundant roles for Syn-3 and Syn-1A pertaining to specific components of

biphasic secretion.

In contrast to Syn-1A which acts specifically on first phase GSIS (Ohara-Imaizumi et al.

2007), our data supports a role for Syn-3 in facilitating both phases of secretion. Although

59

previous studies revealed impaired release of docked and primed insulin vesicles by Syn-1A

depletion, the inhibitory effect was not seen on newcomer granules (Ohara-Imaizumi et al.

2007). Thus, Syn-1A is an unlikely regulator of newcomer granule exocytosis, opening up the

possibility of Syn-3 as the candidate syntaxin in assuming this mechanistic role. Consistent with

this theory, TIRFM of Syn-3 depleted INS-1 showed no change in the number of previously

docked granules, or in the fusion competency of these primed vesicles. This suggests that Syn-3

does not mediate the docking and release of RRP granules underlying first phase GSIS.

However, the exocytosis of newcomer granules recruited from the reserve pool located deeper in

the cytosol also contributes to first phase. These newcomer granules approach the plasma

membrane and are immediately released upon stimulation with minimal docking time (Shibasaki

et al. 2007). We show that Syn-3 regulates these latter events of first phase GSIS as Syn-3

depletion impaired newcomer granule recruitment and fusion in INS-1 cells. Since mobilization

of granules to refill the RRP underlies the entire second phase of GSIS, reduced Syn-3 also

inhibited the sustained release of insulin. Taken together, our data support a role for Syn-3 in

biphasic insulin secretion by accelerating the recruitment of newcomer granules during RRP

replenishment.

In addition to mobilizing newcomer granules, we propose a function for Syn-3 in

regulating the compound exocytosis of insulin vesicles. Syn-3 is the only syntaxin isoform

localized abundantly to insulin granules, reflecting its potential for mediating granule-granule

fusion. If homotypic fusion of insulin granules resembles the process observed in yeast vacuoles

or mammalian endosomes (Hong 2005), it likely involves membrane-anchored SNARE

isoforms. In accordance with the SNARE hypothesis of membrane fusion, Syn-3 must partner

60

with cognate SNAREs to facilitate compound exocytosis. However, it remains uncertain which

isoform of VAMP and SNAP are members of the Syn-3 associated SNARE complex driving

granule-granule fusion in the pancreatic beta cell. Since SNAP-25 redistributes from the plasma

membrane to granule membranes during sequential exocytosis (Takahashi et al. 2004), it likely

participates in the homotypic fusion of vesicles. Based on data from our current study, we

suggest VAMP-2 as the remaining member of the trimeric SNARE complex formation between

insulin granules. VAMP-2 expression was downregulated following Syn-3 knockdown,

indicating a close association between the two SNAREs. Our hypothesis of VAMP-2 as the

cognate partner of Syn-3 could possibly account for the secretion deficits observed in Syn-3 KD

cells. Decreased availability of Syn-3 and VAMP-2 would reduce active Syn-3/SNAP-

25/VAMP-2 complexes, consequently impairing granule-granule fusion and GSIS on a whole.

Aside from decreased VAMP-2, the expression of Syn-2 was also significantly reduced

following knockdown of Syn-3 in INS-1 cells. Presently, the exocytotic role of Syn-2 in the beta

cell remains to be elucidated. Decreased Syn-2 may have contributed to blunted GSIS, but the

mechanism underlying this effect is unknown. Preliminary data from perifusion, IPGTT,

capacitance, and TIRFM studies (Zhu, D., Xie L., Hansen J.B., Gaisano, H.Y., unpublished

observations) suggest an inhibitory role for Syn-2 in secretion. In addition, confocal microscopy

revealed Syn-2 localization to insulin granules upon stimulation with glucose and GLP-1

(unpublished data). These results indicate a potential role for Syn-2 in negatively regulating

granule-granule fusion during compound exocytosis. In accordance with these data, it is

therefore plausible that Syn-2 expression may have been downregulated to compensate for the

impaired secretion of Syn-3 KD cells.

61

Concurrent changes in Syn-2 and -3 are not unusual, and have recently been observed in

Munc18-a/-b double knockdown PC12 cells (Han et al. 2009). These KD cells showed reduced

Syn-2 and -3 levels, suggesting critical roles for Munc18-a and -b in supporting the expression of

both syntaxin isoforms. Of the SM proteins, we believe Munc-18b is the strongest candidate for

regulating the exocytotic role of Syn-3 during GSIS. Munc18-b binds to Syn-3 with the highest

affinity (compared to Syn-1, -2 and -4) (Liu et al. 2007), and has a Syn-3 specific binding site at

the C-terminal end (Liu et al. 2007). This interaction has demonstrated importance in promoting

Syn-3/SNAP-25 formation to allow for fusogenic SNARE complex assembly during epithelial

secretion (Liu et al. 2007). Furthermore, since Munc18-b association with Syn-3 is known to

regulate plasma-granule and granule-granule fusion in mast cells (Tadokoro et al. 2007), we

believe this interaction has similar significance in the pancreatic beta cell. In fact, unpublished

data from Dr. Gaisano’s laboratory demonstrated many of the above postulated actions for

Munc18-b and its interactions with Syn-3 in islet beta cell insulin granule-granule fusion.

Although the mechanism by which Munc18-b regulates Syn-3 in GSIS is undefined, we

suggest Munc18-b may serve as a chaperone to Syn-3 through the secretory pathway, similar to

Munc18-a trafficking of Syn-1 (Arunachalam et al. 2008, Han et al. 2009). With only minor

differences between the Syn-3/Munc18-b complex compared to the Syn-1/Munc18-a complex

(Kauppi, Wohlfahrt & Olkkonen 2002), the coupling of each Syn/Munc18 pair likely involves

similar molecular interactions. Presuming that Syn-3/Munc18-b binding activity is specified by

the conformation of Syn-3, these binding modes would probably resemble those of Syn-

1/Munc18-a interaction. Munc-18a is known to bind Syn-1 in the closed conformation

(Dulubova et al. 1999), which is essential for trafficking Syn-1 through the ER and Golgi

62

complex (Medine et al. 2007). In the same way, it is plausible that Munc18-b may selectively

bind to the closed conformation of Syn-3 in order to traffic to the target membrane. In our study,

open form Syn-3 was mislocalized mainly in a non-insulin granule compartment in the

perinuclear region (likely the Golgi), and less prominent on insulin granules and the plasma

membrane compared to wild-type Syn-3. We suggest Syn-3 open form mutants could not

properly bind Munc18-b, and as a result would not traffic out of this cytoplasmic compartment.

Unable to reach the target site to facilitate membrane fusion, mislocalized Syn-3 mutants would

fail to enhance secretion. This is in fact, consistent with our findings as open form Syn-3 had no

significant effect on GSIS, despite its overexpression in INS-1 cells.

There are likely other proteins that modulate the assembly (or disassembly) of the Syn-3

SNARE complex to carry out its action in exocytosis. For example, Syn-3 function may involve

complexins, grappling proteins which interact with SNARE complexes to regulate GSIS in

pancreatic beta cells (Abderrahmani et al. 2004). Complexin II has been shown to bind isolated

Syn-3 (Tadokoro, Nakanishi & Hirashima 2010), and the Syn-3/SNAP-25/VAMP-2 complex

(Pabst et al. 2000, Pabst et al. 2002). Although complexins have conflicting roles on SNARE-

mediated fusion in neurons, sperm cells and mast cells (Tokumaru 2001, Roggero et al. 2007,

Tadokoro 2005), altered expression of complexin in pancreatic beta cells produced a common

inhibitory effect on vesicle fusion (Abderrahmani et al. 2004). As facilitators of exocytosis,

complexins interact with SNARE complexes in a 1:1 ratio (Chen et al. 2002) to stabilize its

highly fusogenic state immediately before release (Reim et al. 2001, Xue et al. 2007). From our

data, we suggest that decreased Syn-3 and VAMP-2 expression in our KD cells may have led to

corresponding reductions in Syn-3/SNAP-25/VAMP-2 complexes available to bind complexins.

63

This imbalance may have perturbed the expression and activity of complexins, and based on

previous reports, is predicted to inhibit GSIS in pancreatic beta cells.

4.2 Experimental Limitations

Although Syn-3 overexpression in HIT cells has been reported to inhibit insulin secretion

(Kang et al. 2002), we believe the effects of syntaxin upregulation often depend on the beta cell

line chosen for study. Such is the case with Syn-1A, as overexpression decreased insulin

biosynthesis and content in HIT cells (Kang et al. 2002) and a Syn-1A overexpressing mouse

(Lam et al. 2005), but increased insulin content in the βTC3 cell line (Nagamatsu et al. 1996).

These differences indicate the complexity of syntaxin regulation on GSIS and demonstrate how

various aspects of the insulin secretory process may be altered. These effects include changes to

the SNARE exocytotic machinery, ion channel activity and insulin gene transcription and

biosynthesis. Certain effects may be more pronounced in particular beta cell lines, accounting

for the observed discrepancies in overexpression studies. It is therefore important to conduct

similar experiments on primary cells to supplement data obtained from cell lines. By

overexpressing Syn-3 in mouse islets and observing enhanced secretion, we confirmed that Syn-

3 potentiation of GSIS is not limited to only the INS-1 cell line. Conversely, we performed

siRNA experiments (acutely and stable knockdown) on INS-1 cells to reduce insulin secretion,

and will have to confirm these results in lenti-shRNA knockdown of Syn-3 in pancreatic islets,

the latter to examine reduction in biphasic insulin secretion,

64

We acknowledge another limitation to our study is in the heterogeneity of the Syn-3 KD

cell line which may have concealed possible changes in the expression of other exocytotic

proteins. Despite anticipated reductions in Munc18 proteins known to interact with Syn-3, no

differences were observed in KD versus control. Given the strength of evidence showing the

closely linked expression of syntaxin and SM proteins (Verhage et al. 2000, Lam et al. 2005,

Voets et al. 2001, Toonen et al. 2005), modest reductions in Munc18 were likely masked by the

presence of incomplete knockdown cells. For a more accurate assessment of protein expression

after Syn-3 knockdown, a pure cell line should be isolated through colony selection and then re-

examined for potential compensation effects. A clean Syn-3 KD model characterized by

pronounced secretion deficits would confirm the critical function of Syn-3 during GSIS, and also

reveal any changes to protein expression that may have been previously overshadowed.

4.3 Future Directions

The sensitivity of GSIS to syntaxin expression has been widely acknowledged in Syn-1A

overexpression and knockdown studies performed in islets and insulinoma cell lines (Nagamatsu

et al. 1999, Lam et al. 2005, Nagamatsu et al. 1996). Interestingly, both forms of Syn-1A

perturbation produced severe deficits in GSIS, suggesting syntaxin regulated fusion may follow a

bell shaped curve function. Overexpressed Syn-1A may form incomplete SNARE complexes

that may inhibit or compete with fusogenic SNARE complexes, or excess Syn-1A may directly

affect ion channels that participate in inhibiting the secretory process (Leung et al. 2007).

According to this theory, optimal secretion would be achieved within a specified range of

65

syntaxin expression that orchestrates the exocytotic and ion channel events (Leung et al. 2007).

Fluctuations in endogenous syntaxin levels are proposed to inhibit ion channel activity and

exocytotic machinery, thereby impairing secretion. While Syn-3 is less likely to have such

diverse influence in the different components of the insulin secretory process, it remains a

possibility, particularly with calcium channels (see below). To determine whether this principle

is applicable to Syn-3, we should measure the dose dependent effects of Syn-3 on GSIS and ion

channel activities. The obtained dose-response curve would then elucidate the sensitivity of Syn-

3 expression on these secretory components and further support their essential role in

contributing to GSIS in pancreatic beta cells.

While we have established a function for Syn-3 in the recruitment of newcomer granules,

the molecular interactions underlying this process remain uncertain. To more clearly examine

Syn-3 mediated granule dynamics, we can use electron microscopy to provide details on granule

size and number during conditions of elevated or depleted Syn-3 expression. In addition, our

finding that Syn-3 acts specifically on newcomer granules can be strengthened with

electrophysiological studies. Single beta cell exocytosis can be analyzed with membrane

capacitance measurements using a series of depolarizations to stimulate secretion. A series of

pulses sequentially triggers the release of primed RRP vesicles, followed by the exocytosis of

newcomer granules. We anticipate altered Syn-3 expression to amplify or attenuate capacitance

increases triggered by the later pulses. This would correspond to changes in the fusion of

newcomer granules and complement our findings from this study.

66

We have previously reported Syn-3 to influence L-type Ca2+ channel activity (Kang et al.

2002), and this effect is worth re-examining in INS-1 cells. Using patch clamp techniques we

can measure changes in calcium current following perturbation of Syn-3 expression. Since Syn-

3 regulates biphasic GSIS, we aim to examine the activity of L-type Ca2+ channels, known to

trigger first phase release, and R-type Ca2+ channels which control the Ca2+ influx required for

RRP replenishment during second phase GSIS (Schulla et al. 2003, Jing et al. 2005).

Interestingly, recent research has proposed newcomer insulin granules to exhibit higher calcium

sensitivity compared to the RRP (Pedersen, Sherman 2009). According to their theory, this

highly Ca2+ -sensitive pool of granules (HSCP) responds to cytosolic calcium concentrations

mediated by R-type Ca2+ channels. Therefore, if we confirm an influential role of Syn-3 on R-

type Ca2+ channels activity, it would suggest that Syn-3 regulation of newcomer granule

exocytosis involves changes to the release rate of the HSCP.

Our final goal for future experiments is to characterize the protein interactions of open

form and wild type Syn-3. By performing pull-down assays, we can show how Syn-3

conformation may dictate its binding activity with other regulators of exocytosis. Proteins found

consistently bound to Syn-3 imply these interactions may be important for mediating the role of

Syn-3 during GSIS. We can also quantify binding affinity in order to determine whether

exocytotic proteins, such as Munc18 and complexin, selectively interact with Syn-3 in a specific

conformation. More comprehensive data on Syn-3 binding modes can supplement our current

findings and provide insight into the differential effects of wild type versus open form Syn-3 on

GSIS.

67

4.4 Physiological Relevance of Syntaxin-3 Function in Glucose Stimulated Insulin Secretion

Glucose stimulated insulin secretion in pancreatic beta cells was completely abolished

following neurotoxin mediated cleavage of VAMP-2 (Regazzi et al. 1995). However, when

similar conditions produced a loss of Syn-1 function, insulin release was impaired by only 25%

(Yang et al. 1999, Land et al. 1997). These results revealed that VAMP-2 was indispensable to

secretion, while Syn-1 appeared to regulate GSIS alongside other syntaxin isoforms. This is in

fact, demonstrated by our study which found Syn-3 to facilitate exocytosis of newcomer granules

during biphasic GSIS. Prior to this report, the exocytotic role of Syn-3 in the pancreatic beta cell

was undetermined. By confirming a function for Syn-3 in biphasic GSIS, we have shown that

both phases of secretion are regulated by multiple syntaxin isoforms. Syn-3 supports first phase

secretion alongside Syn-1, but acts specifically on RRP replenishment which is differentiated

from Syn-1 control of docked granules (Ohara-Imaizumi et al. 2007). Syn-3 regulation of

second phase GSIS is also significant as previously Syn-4 was the only syntaxin implicated in

this phase (Spurlin, Thurmond 2006).

The presence of numerous syntaxin isoforms in the beta cell may function to dictate the

compartmental specificity of insulin granule exocytosis. Pairing of VAMP-2 with distinct

syntaxin isoforms is proposed to differentiate between granule-granule vs. PM-granule fusion

events. Furthermore, the ability of Syn-3 to influence calcium channel activity may reveal a

mechanism which specifies the exocytosis of newcomer granules as opposed to those residing in

the RRP. By elucidating the role of Syn-3 in vesicle targeting and release, we can gain a more

68

detailed understanding of the granule dynamics underlying sequential vs. compound exocytosis

during glucose stimulated insulin secretion.

69

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