Biochemical and cellular analysis of mouse Srv2/CAP (cyclase associated protein)

in regulating actin dynamics

Senior Thesis

Presented to

The Faculty of the School of Arts and Sciences

Brandeis University

Undergraduate Program in Biology

Dr. Bruce Goode, Advisor

In partial fulfillment of the requirements for the degree of Bachelor of Science

by

Leslie Golden

May 2013

Copyright by

Leslie Golden

Committee members:

Name: ____Dr. Bruce Goode___________Signature:_____________________________

Name: ___Dr. Satoshi Yoshida________Signature:_____________________________

Name: ____Dr. Daniela Nicastro________Signature:_____________________________

Abstract

Srv2/CAP is a highly conserved actin binding protein. Extensive previous

analysis has shown that yeast Srv2/CAP increases cellular actin dynamics by both

stimulating cofilin-mediated severing of actin filaments (an activity mediated by its N-

terminus) and by catalyzing the conversion of actin monomers from an ADP- to ATP-

bound state (an activity mediated by its C-terminus). On the other hand, the regulatory

activities of mammalian CAP have remained largely uncharacterized. We show here that

the activities of mouse CAP1 are indistinguishable from those of yeast Srv2. First, in

biochemical assays CAP1 strongly enhanced actin filament disassembly by cofilin, and

stimulated nucleotide exchange (ADP for ATP) on actin monomers. Second, these

activities depended on specific sequences in CAP1 that are shared with Srv2 and found to

mediate the same activities in Srv2. Third, expression of CAP1 could fully rescue srv2!

defects in yeast cell growth and actin organization. These observations demonstrate that

both the biochemical and cellular activities of Srv2 in regulating actin dynamics are

highly conserved from yeast of mammals

Introduction

Actin is involved in many important cellular processes including division, motility,

gene transcription, and cell polarity, among others. Disruptions to the dynamics of the

actin cytoskeleton can be associated with cancer, compromised immunity, and

neurodegenerative diseases (Carnell and Insall, 2011).

The turnover of actin monomers enables actin filament dynamics. In actin

turnover, ATP-G-actin (globular actin) monomers add onto the barbed end of actin

filaments, becoming ATP-bound filamentous actin (ATP-F-actin). While part of the

filaments, F-actin hydrolyzes its bound ATP to ADP, becoming ADP-F-actin. ADP-F-

actin can dissociate from the actin filament at the pointed end, becoming G-actin again.

Nucleotide exchange (ADP to ATP) occurs on the actin monomer before the monomer

can be added to another actin filament. However, actin turnover is inherently slow, and

requires the addition of other components to speed it up. There are about 30 proteins

regulating the disassembly and assembly of actin filaments (Mosely and Goode, 2006).

These proteins include formins and profilins, which are involved in actin assembly, as

well as cofilin, AIP1, coronin, and Srv2/CAP, which are involved in actin disassembly.

Srv2/CAP

The Srv2 protein was initially discovered as a mutant suppressor allele of RAS in

yeast. Srv2 was shown to stimulate the production of cAMP subsequent to the activation

of RAS. (Fedor-Chaiken et al, 1990). CAP is the mammalian isoform of yeast Srv2.

Yeast Srv2 function in vivo

In the actin cytoskeleton, Srv2 is involved in actin turnover by assisting cofilin-

mediated actin severing. Knock down of Srv2 expression in yeast cells causes yeast to be

larger and rounder than wild type yeast, show severe depolarization of cortical actin

patches, have reduced actin cable staining, and impaired growth at 37°C. The function of

Srv2/CAP was examined using genetic crosses with Tropomyosin 1 (tpm1), a protein that

binds to and protects actin filaments. srv2! was shown to rescue a tpm1! phenotype

(Chaudhry et al., 2013), a result also found in genetic crosses of aip1! and tpm1! (Okada

et al., 2006). The ability of srv2! and aip1! to rescue a tpm1! phenotype implies that

AIP1 and Srv2 have similar functions, and as AIP1 is involved in disassembly of actin

filaments, this indicates that Srv2/CAP may be involved in this as well.

Mammalian CAP1 function in vivo

There are two isoforms of CAP expressed in mammals, CAP1 and CAP2. CAP1

is ubiquitously expressed, whereas CAP2 appears to be muscle specific (Peche et al.,

2007). Both CAP proteins share a reasonably high degree of sequence homology with

their Srv2/CAP counterparts in other species (30-40% identity from yeast to humans)

(Peche et al., 2007).

CAP1 in expressed in all tissues except skeletal muscle. In the brain, Northern

blot analysis showed especially high CAP1 expression in the hippocampus, as well as the

cortex and striatum. In mammalian cell cultures, CAP1 generally colocalized with

cofilin-1 and actin to the leading edge of the lamellipodia, but it did not colocalize with

cofilin in the nucleus. CAP1 was also diffusely present throughout the cytoplasm

(Bertling et al., 2004).

CAP1 has been knocked down by RNA interference in the mammalian cell lines

NIH3T3, B16F1, and Neuro 2A. Silencing led to depolarized actin networks, and

disrupted cofilin localization, moving form the leading edge to punctate cytoplasmic

structures. (Bertling et al, 2004). This indicates that CAP1 is necessary for correct

cofilin-1 localization. CAP1 knockdown cells have additional, thicker stress fibers that

are aligned in parallel; most of the F-actin within these cells is accumulated into the stress

fibers (Bertling et al, 2004). The stress fibers in the CAP1 knockdown cells also showed

a reduced rate of actin filament turnover, implying that the fibers become more resistant

to depolymerization in the absence of CAP1. These observations suggest that Srv2/CAP

helps promote actin filament disassembly and turnover in vivo (Bertling et al, 2004).

Motility assays showed that the CAP1 depletion also reduced cell motility to one

half the rate observed for wild-type cells (Bertling et al, 2004). This indicates that the

presence of CAP1 is essential for the maintenance of the actin dynamics that enable

normal cell mobility.

CAP1 overexpression has been implicated in pancreatic cancer tissues, and

statistical analysis of pancreatic cancer patient data indicated that CAP1 overexpression

is associated with neural invasion, resection margin, lymph node metastasis and a

generally poor prognosis. CAP1 knockdown in these cells resulted in reduced formation

of lamellipodia and reduced cell motility, as tested by a transwell-chamber motility assay

(Yamazaki et al, 2009). Altogether, this strongly suggests that CAP1 plays a role in cell

movement in normal and neoplastic cells (Yamazaki et al, 2009).

Mammalian CAP2 function in vivo

CAP2 is the muscle specific CAP isoform, and is primarily present in the heart,

skeletal muscle, all layers of the skin (Peche et al, 2007), and specific parts of the brain,

including the cortex, striatum, and hippocampus (Bertling et al, 2004). CAP2 is also

weakly expressed in the liver and lungs (Bertling et al, 2004). Biochemical assays with

CAP2 show that its C-terminus interacts with monomeric actin to inhibit the formation of

actin gels, implying that it binds actin monomers like other CAP proteins (Peche et al,

2007).

CAP2 shows various localization patterns depending on the cell type. In

embryonic cardiomyocytes, CAP2 has been shown be present in both the cytoplasm, and

the nucleus. In non-differentiated myogenic cells, CAP2 was primarily present in the

nucleus, but upon differentiation, it diffused throughout the cytoplasm. In cardiac muscle

cells and mouse skin cells CAP2 was localized in the nucleus (Peche et al, 2007). This

implies that CAP2 may change its location in cardiac cells during their development.

The knockdown of CAP2 expression leads to cardiomyopathy (a disease

involving variations in heart muscle thickness) in mice (Peche et al., 2012). However,

CAP2 deficiency did not affect embryo size, meaning that it manifested itself

phenotypically shortly after birth. Mice lacking CAP2 were smaller than their wild-type

counterparts – weighing 30-45% less – and died earlier, possibly due to the abnormalities

in their hearts. Mutant mice had much larger hearts, caused by severe dilation of all four

chambers, especially the ventricles and the right atrium. Although the chambers were

enlarged, the ventricular myocardium was thinner in CAP2 knockdown mice than in their

wild-type littermates, and these mice had a slower heart rate. Apoptosis of cardiac cells

was also greater in mutant mice (Peche et al, 2012), possibly accounting for their cardiac

defects.

CAP2 expression was also knocked down in zebrafish, but these mutant embryos

died before they were born. Unlike the knockdown of CAP2 expression in mice, the

CAP2 knockdown in zebrafish manifests itself before birth, and is lethal. Zebrafish

embryos lacking CAP2 are shorter, and display pericardial edema, further implying a role

for CAP2 in heart development. They have almost no motility. Double knockdown with

CAP1 was also performed, but it showed no increase in the number of short zebrafish

embryos, implying that CAP1 does not rescue a CAP2 knockdown (Effendi et al, 2012).

Up-regulation of CAP2 has been shown in bladder tumors, colon tumors, thyroid

tumors, kidney tumors (it is completely absent in normal kidneys), and brain tumors.

Down-regulation of CAP2 has been demonstrated in breast cancer tumors (Peche et al,

2007). CAP2 has also been shown to be upregulated in hepatocarcinomas (HCC). In

HCC cell lines, CAP2 was present in the perinuclear area, and, in highly metastatic HCC

cells, it colocalized with actin at the leading edge of lamellipodia. CAP2 knockdown

decreased the amount of migrating cells, while CAP2 overexpression was generally

observed in progressed HCC, and was significantly correlated with tumor size, poor

differentiation, portal vein invasion, and metastasis (Effendi et al, 2012).

Structure

CAP is a multidomain protein that can be functionally divided into an N-terminal

and a C-terminal half that are involved in different steps of actin cytoskeleton dynamics.

Figure 1: Domains of Srv2/CAP.

The N-terminus of CAP

The N-terminus of CAP includes the helical folded domain (HFD), which is

single folded domain comprised of a bundle of multiple a-helices that dimerize in

solution (Mavoungou et al, 2003; Yusof et al, 2005). The HFD’s contribution to actin

function was investigated in our lab using mutagenesis to create five alleles (Srv2-90 to

Srv2-94), in which 2-3 conserved, solvent-exposed residues in each patch were replaced

with alanines. These mutants (in the context of the intact protein/gene) were tested for

their ability to rescue defects in cell growth and actin organization caused by srv2!. Two

mutations, srv2-90 and srv2-91, failed to rescue defects. These mutant yeast were

abnormally round and large, showed abnormal actin cytoskeleton organization, and were

impaired in growth at elevated temperatures compared to wild type yeast.

Immunofluorescence microscopy demonstrated that these mutant srv2 alleles colocalized

with actin similar to wild type Srv2 protein, indicating that these alleles may alter Srv2

activities and interactions, rather than localization. Genetic crosses were also performed

between the srv2 alleles and alleles of profilin (pfy1-4) and cofilin (cof1-19) that are

impaired in actin binding. Both pfy1-4 and cof 1-19 were synthetic lethal when crossed

with srv2! yeast. srv2-90 and srv2-91 also displayed synthetic lethality with pfy1-4 and

cof1-19 (Quintero-Monzon et al, 2009), suggesting that the conserved surface residues

targeted in srv2-90 and srv2-91 form a critical functional site in the HFD that is integral

to Srv2 in vivo function.

Biochemical assays support these results; purified srv2-90 and srv2-91 proteins

showed decreased actin turnover in phosphate release assays, and were unable to pull

down the cofilin-actin complex in a supernatant depletion assay (Quintero-Monzon et al,

2009). This implies that the srv2-90 and srv2-91 alleles have mutations that change their

cofilin-actin binding sites, thus significantly inhibiting the function of NSrv2.

Furthermore, disassembly assays showed that purified full-length Srv2 in combination

with cofilin stimulate actin filament severing, whereas as srv2-91 does not. When tested

in conjunction with cof1-9, a mutant with a disrupted HFD binding site, Srv2

enhancement of cofilin-mediated actin disassembly was disrupted. TIRF microscopy

revealed that NSrv2 (and full-length Srv2) enhanced the efficiency of filament severing

by cofilin (Chaudhry et al, 2013). Thus, the HFD of Srv2 binds to cofilin on F-actin and

enhances filament severing to promote actin disassembly.

The activities of full length mammalian CAP1 in regulating cofilin-mediated actin

disassembly were recently explored by Normoyle and Brieher (2012). Their results

demonstrate that CAP1 acts on the level of the actin filament to increase cofilin-mediated

actin severing events. It was also demonstrated that CAP1 alone is capable of severing

actin filaments at acidic pH, but its severing activity decreases as the pH increases

(Normoyle and Brieher, 2012). Cofilin activity is also pH dependent, and is most active

in severing filaments at a basic pH (Rottenberg et al, 1985). This means that throughout

the physiological pH range, 6.8-7.4, only the combination of CAP1 and cofilin will

provide strong severing activity (Normoyle and Brieher, 2012).

The first 50 amino acids of CAP form as segment that has been referred to as the

coiled coil (CC) domain, even though this sequence has a relatively low prediction value

for forming a coiled coil. H-NMR analysis has also suggested that the ‘CC’ region of

CAP has an unstructured and flexible arrangement, inconsistent with a coiled coil

(Mavoungou et al, 2003).

Regardless of what structure it forms, the CC domain has been shown to be

required for yeast Srv2 oligomerization into hexamers. Using hydrodynamic analysis,

the oligomerization state of full-length Srv2 was shown to be hexameric (Chaudhry et al.,

2013), whereas Srv2!CC was dimeric (Quintero-Monzon et al., 2009). Further,

Chaudhry et al. (2013) used electron microscopy and single particle analysis to solve the

structure of the NSrv2 (which included the CC and HFD domains) at ~20 Angstrom

resolution, revealing that NSrv2 forms hexamers with six symmetrical protrusions,

resembling ninja stars, or ‘shurikens’ (Chaudhry et al., 2013). Further, Srv2!CC only

partially rescued an srv2! mutation in vivo (Quintero-Monzon et al., 2009; Chaudhry et

al., 2013). Suggesting that formation of hexamers by Srv2/CAP critical for its actin

regulatory functions both in vivo.

The C-terminus of CAP

The main domain in component of CSrv2 is an elliptical barrel composed of 6

coils, each formed by three right-handed parallel !-sheets, and thus called the !-sheet

domain (Dodatko et al., 2004).

Almost 20 years ago, the in vivo function of the "-sheet domain was dissected

with 10 mutant alleles (Srv2-103 to Srv2-112). Three mutant alleles, srv2-104, srv2-108,

and srv2-109 failed to rescue an srv2! phenotype. srv2-104 resides on the surface of the

B-sheet, while srv2-108 and Srv2-109 reside in connecting loops. srv2-104 yeast

displayed enlarged mother cells and small daughter cells, reduced actin staining and a

depolarized actin network, and a higher frequency of multi-budded cells in comparison to

wild type cells. srv2-108 and srv2-109 also showed large, round cells, and a depolarized

actin network, and in addition they showed elongated buds (Mattila et al., 2004).

In biochemical assays, the mutant Srv2 proteins, srv2-104, srv2-108, and srv2-109

displayed reduced affinity for ADP-G-actin in contrast to the wild-type protein. The

srv2-104 mutant was also tested for its affinity to ATP-G-actin and showed similar

affinity to ATP-G-actin as the wild type protein. Thus, these mutations disrupt key ADP-

G-actin binding sites in Srv2, implying that the "-sheet is required for Srv2 binding to

ADP-G-actin (Mattila et al., 2004).

On the N-terminal side of the "-sheet domain, in between two proline rich

sequences (P1 and P2) and an intervening WASp-homology 2 (WH2) domain. To

explore the functional importance of the WH2 domain, Chaudhry et al. (2010) designed

four alanine substitution alleles (srv2-96 to srv2-99) targeting conserved residues. The

functionality of these alleles was measured based on their ability to rescue srv2!

phenotypes. One allele in particular, srv2-98, in which all four residues of a conserved

LKKV motif were replaced by alanines, had the strongest defects, almost as bad as

srv2!. srv2-97 also showed morphological defects, although they were not as

pronounced as srv2-98. In biochemical assays testing the ability of these mutant proteins

to suppress actin filament assembly, srv2-98 had strongly impaired actin monomer

binding, while srv2-97 displayed only slight defects (Chaudhry et al., 2010). This

conserved motif is thus binds actin monomers and is critical for the WH2 domain’s

function in Srv2. Further, the WH2 domain could bind ATP- and ADP-actin monomers

equally well.

To better understand the contribution of the WH2 domain to CAP function,

nucleotide exchange assays (on cofilin bound ADP-actin monomers) were performed

with full length mutant Srv2 proteins, srv2-97 and srv2-98. The mutants displayed close

to no activity, indicating that the WH2 domain is required for the nucleotide exchange

function of Srv2/CAP. Further analysis examining the nucleotide exchange capabilities

of the different domains of Srv2/CAP indicated that while the WH2 domain is necessary

for this function, it also requires the "-sheet domain (Chaudhry et al., 2010).

One of the two proline rich regions (P1) surrounding the WH2 domain of Srv2

binds profilin (Witke et al., 2004, Bertling et al., 2007). In contrast, the P2 region binds

to the SH3 domain of Abp1 to help localize Srv2 to the cortical actin structure in vivo

(Freeman et al., 1996). ). Profilin and Srv2 do not compete with each other for actin

binding to actin, and Srv2 can simultaneously bind profilin and actin monomers. Genetic

analysis with a mutation that disrupts profilin-binding, srv2-201, showed that srv2-201

partially rescues the srv2! phenotype (Bertling et al., 2007). In addition, srv2-201 was

able to partially rescue pfy1-4 defects, suggesting a functional interplay between Srv2 and

profilin in vivo, possibly in recycling actin monomers from the ADP- to ATP-bound state

in preparation for new rounds of assembly.

The second proline rich (P2) region of Srv2 is very similar to the consensus

sequence of SH3 domains, implying that Srv2 binds to SH3 domains of several proteins,

including Abp1 (actin binding protein 1). Srv2-Abp1 interactions were demonstrated by

their co-immunoprecipitation from cell lysates and in direct binding assays in vitro using

purified proteins (Freeman et al., 1996). Localization of Srv2 to cortical actin patches in

yeast cells was also shown to be dependent on interactions of the Srv2 P2 region with the

SH3 domain of Abp1 (Freeman et al., 1996).

Together, these studies show that the C-terminal half of Srv2/CAP

(CSrv2) contains two different actin monomer binding sites, a WH2 domain that equally

well to ATP- and ADP-actin, and a B-sheet domain that strongly prefers binding ADP-

actin monomers; and both sites contribute to CSrv2 biochemical and cellular functions.

Further, there is a profilin-binding site (P1), adjacent to the WH2 domain, which

facilitates the coordination of Srv2 and profilin effects in recycling actin monomers, and

there is an SH3 domain-binding site (P2) that mediates Abp1-dependent localization of

Srv2 to cortical actin sites in vivo.

To summarize, the C-terminal half of Srv2 localizes the protein in vivo and

facilitates actin monomer processing, whereas the N-terminal half of Srv2 enhances

cofilin-mediated severing and disassembly of filaments through formation of hexameric

shuriken structures.

Conclusion and Unanswered Questions

There are two rate limiting steps in actin filament turnover: dissociation of actin

subunits from filaments ends, and nucleotide exchange (ADP for ATP) on actin

monomers. Srv2/CAP catalyzes both steps using the separate activities of its two halves

(NSrv2 and CSrv2, respectively). The N-terminus of Srv2/CAP enhances filament

disassembly by increasing the efficiency of cofilin severing of filaments, and the C-

terminus of Srv2/CAP catalyzes dissociation of cofilin from ADP-actin monomers, and

nucleotide exchange to recharge monomers (back to an ATP-bound state) for new rounds

of assembly. However, virtually all of this mechanistic work has been done on yeast

Srv2, leaving it uncertain whether the mammalian homologue, CAP1, has similar

functional activities and/or mechanisms. My thesis research has addressed this question,

through both in vivo and in vitro tests. In vivo, I have tested the ability of wild type and

mutant CAP1 constructs to ‘rescue’ in vivo defects in cell growth and actin organization

caused by srv2!. In vitro, I am have purified CAP1 (full length and fragments) and

investigated whether they have similar or distinct biochemical activities on actin

turnover, as well as dissected their mechanisms by introducing mutations analogous to

those used in the studies described above on yeast Srv2.

My results indicate that CAP1 is capable of rescuing the perturbed actin network

srv2! yeast in vivo, and that it is capable of performing in vitro like Srv2. We show that

NCAP1 enhances cofilin mediated actin severing, and that CCAP1 is capable or

recharging actin monomers. In addition, we isolate these functions to conserved, surface

exposed residues of CAP1 that are analogous to functionally important residues in Srv2.

Materials and Methods

Cloning of the CAP1 wild type and mutant plasmids

A CAP1 integration plasmid was created by placing the CAP1 open reading frame

into FC01, a URA-marked CEN plasmid containing the promoter of Srv2, at the NotI and

BamHI restriction enzyme sites. The CAP1 insert was created by PCR (5µL pfu buffer,

4µL dNTPs, 2µL MgSO4, 1µL template DNA, 1µL sense primer, 1µL antisense primer,

35µL MQ, 1µL pfu).

The CAP1 product underwent PCR clean up and was eluted in 32µL MQ. This

CAP1 insert, and the FC01 empty vector were digested with NotI and BamHI for 2hrs at

37°C. The digested DNA was run on a 1.2% agarose gel; the appropriate bands were cut

out and the DNA was retrieved by gel purification. The purified CAP1 insert and FC01

vector were ligated at room temperature for 2 hrs.

The FC01_CAP1 plasmid was transformed into XL1-BLUE bacteria. The bacteria

were left on ice for 30min, heat shocked in a 42°C water bath for 45 sec, and placed on

ice for 2 min. 500µL of Luria broth (here on referred to as LB) was added, and the tubes

were rotated at 37°C for 45 min, and then plated on LB/AMP plates and grown overnight

at 37°C. Next day, single colonies were picked and grown in 5mL LB/AMP media,

rotating overnight at 37°C. DNA was obtained from these bacterial cultures by mini-prep

and analyzed by restriction digest for the presence of the CAP1 insert. Positive clones

were confirmed by sequencing.

CAP1 mutants were generated using the QuickChange protocol (Stratagene).

Briefly, primers containing the desired mutation flanked by a silent mutation creating a

new restriction site were phosphorylated by adding 5µL primer, 1µL PNK, 1µL PNK

buffer, and 3µL of MQ and incubating at 37C for 1 hr. The mutation was generated in

the FC01_CAP1 plasmid by PCR (5µL pfu buffer, 4µL dNTPs, 2µL MgSO4, 100ng

template DNA, 1µL phosphorylated primer, 36µL MQ, 1µL pfu). The PCR product was

digested for 1 hour with 1µL Dpn1 at 37°C. A PCR cleanup was performed on the

digested product, and the DNA was eluted in 30µL MQ. The plasmid containing the

mutation was transformed into XL1-BLUE cells according to the same protocol as above.

After the XL1-BLUE cells were grown overnight at 37°C, 10-20 colonies were

picked for a 5mL culture in LB/AMP at 37°C miniprepped according to the protocol

above and screened by restriction digest for the new restriction site.

Yeast transformations

All yeast transformations were performed according to the following protocol

(Goode et al., 2000). Yeast from glycerol stocks were plated on YPD and grown

overnight at 25°C. Using sterile technique, serial dilutions of the yeast were grown in 5

mL of YPD media rotating overnight at 25°C. Next day, cells were harvested and

resuspended in 100µl of TE/0.1M LiOAc and the following was added in this order: 10

µl salmon sperm DNA (10mg/ml ), that was freshly boiled for 2-5 min. and then placed

on ice for 5 min. before use; 2-5µl of miniprepped plasmid DNA, and 750 µl of buffer

(75 µL 10X TE, 75 µL 1M LiOAc/ 600 µL 50% PEG-3350 per transformation) made

fresh immediately before use. The yeast were rotated overnight at 25°C. Subsequently,

the cells were pelleted, resuspended in 100 µL ddH2O and plated on drop-out Leu, Trp,

or Ura plates.

After 2 or 3 days, the yeast colonies were restruck on their respective drop-out

media and used for subsequent experiments.

Crossing Strains

Strains, one mata and one mat#, were streaked together on drop-out plates and

grown at 25°C. A 2mL yeast culture was grown in YPD at 25°C overnight. The next

day, 1mL was spun down and resuspended in 3mL SPO media. The SPO culture was

rotated for 5-10 days at 25°C.

After this time, 45µL of the yeast culture was spun down and rinsed twice with

ddH2O. The yeast were then resuspended in 45µL sterile ddH2O. To this was added 5µL

zymolase, and the yeast were digested for 8-10 minutes at room temperature. Using a cut

tip, 12µL of yeast were pipetted in a straight line onto a YPD plate. Tetrads were

dissected.

Genotyping tetrads was done by stamping the tetrads onto different drop-out

plates and analyzing which tetrads grew, and which died. Yeast tetrads that grew on the

appropriate media were streaked out on drop-out media and used in further experiments.

Actin cable staining

Serial dilutions of 5mL yeast cultures were grown at 25°C. Yeast in YPD were

grown to an OD of 0.2-0.6; yeast in drop-out media were grown to an OD of 0.08-0.2.

To fix the cells, 455µL formaldehyde was added to the 5mL yeast cultures, and rotated at

25°C for 30-35 minutes. The yeast were spun down in 15mL conical tubes (2500rpm, 2

min). The supernatant was decanted and the yeast were washed three times in 1mL of 1x

PBS. The washed cells were stored in PBS, and kept on ice up until 4 weeks after

fixation.

Different concentrations of fixed cells, and a PBS control were stained with

Alexa488-phalloiden (Invitrogen) for 2 days at 4°C in the dark. The cells were then

washed 3 times with 1mL 1X PBS and resuspended in 20µL 1x PBS. Cells were

mounted onto slides and imaged using the FITC setting on a Zeiss E600 microscope

(Thronwood, NY) outfitted with a Hammatsu Orca ER CCD camera (Bridgewater, NJ)

running the Openlab program.

Cell growth

After growing in a 5mL yeast culture, 1 OD of yeast was spun down and

resuspended in 150µL of ddH2O. This was serially diluted (15µL of cells in 135µL

ddH2O) and plated on YPD plates. Plates were grown at 25°C, 30°C, 35°C, and 37°C.

Protein Purification

N-terminal his-tagged CAP1 proteins were E. Coli. First, a 100mL starter culture

was grown overnight at 37°C. Next day, the culture was diluted in 1L LB, grown to

OD600 0.8-1 and induced overnight at 18°C with 0.4 µM IPTG.

The cells were spun down and scraped into a 50mL conical tube and froze in

liquid nitrogen. The cells were thawed and resuspended in 40mL protein buffer (50mM

PBS pH 7.4, 300mM NaCl, 1mM DTT, 10mM imidazole) and protease inhibitors

(PMSF, Aqpi, PepA). The cells were lysed by sonication, and spun (14,000rpm, 20 min).

The supernatent was applied to 1mL of nickel-nitrotriacetic acid-agarose resin (Qiagen,

Valencia, CA) and rotated for 60-90 minutes at 4°C.

The beads were transferred to a column and washed 3 times with 5 column

volumes wash buffer (protein buffer supplemented with 40mM imidazole). The protein

was eluted in 7 seven column volumes of elution buffer (protein buffer supplemented

with 250mM imidazole), one column volume at a time.

The eluted protein was monitored on a 12.5% acrylamide gel and stained with

coomassie.

Actin disassembly assays

Monomeric rabbit muscle actin was incorporated with pyrene actin into

actin filaments. The resulting filaments were made up of 10% pyrene actin (2.7µM

RMA, 0.3µM pyRMA). The calcium buffering the rabbit muscle actin was exchanged

for magnesium by incubating the 10% pyrene labeled actin with Mg EDTA for 2 min at

room temperature. Actin filament formation was initiated by the addition of a high salt

solution (20x initiation mixture) and F-buffer. This solution was kept for 2 hrs at room

temperature in the dark to enable to filaments to form.

The ability of proteins to disassembly F-actin was determined by measuring the

fluorescence of the actin in the presence of these proteins. To a cuvette was added 40µL

of F-actin, with a cut tip, with 10µL of F-buffer, and this was incubated for 5 min. To

this was added varying amounts of CAP1 constructs and HsCof1. Disassembly was

initiated by the addition of 2µL Vitamin-D binding protein (Sigma Aldrich) and 2µL of

3µM CapZ. The rate of disassembly of actin filaments was measured using a PTI.

Nucleotide exchange assays

CAP1’s ability to perform nucleotide exchange on actin monomers was measured

by measuring the fluorescence of etheno-ATP (Molecular Probes, Eugene, OR). G-actin

mixture (actin, hexokinase beads, glucose, ADP) was rotated overnight at 4C. The actin

and various proteins were mixed and added to ethano-ATP and measured.

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-#'(.<*,%+&A(.-0%*&.*=%-)0<*/$"#&0%<*-.)*6@8*>-0*3&'-'%)*&B%3.(/,'*-'*CDE:***

Results

Mouse CAP1 expression rescue the actin organization defects of srv2! yeast

Srv2/CAP proteins are conserved from yeast to mammals and have been shown to

be important for normal actin organization in cells. More specifically, the yeast

homologue Srv2 was shown to be involved in actin disassembly by stimulating severing

of actin filaments by cofilin, as well as recharging released actin monomers for

incorporation in growing filaments. To study the conservation of these functions in the

mouse homologue, CAP1, we first took an in vivo approach.

Mutant srv2! cells are unusually large and round, display depolarized actin

patches, and decreased actin cable staining. However, the srv2! phenotype can be

rescued by transformation with a low copy SRV2 plasmid. We investigated whether

CAP1 is also capable of rescuing the srv2! phenotype. For this purpose, we constructed

a CAP1 integration plasmid and transformed this into srv2! cells. The SRV2 plasmid

was used as a positive control while an empty plasmid served as a negative control.

First, we analyzed cell growth by plating serial dilutions of the yeast strains on

rich medium (YPD) plates, and growing them at different temperatures. Consistent with

published results (Quintero-Monzon et al., 2009), the SRV2 plasmid rescued the growth

defects of srv2! at higher temperatures (34°C and 37°C). Similarly, the CAP1 plasmid

almost restored cell growth at 34°C, albeit less completely at 37°C.

Deletion of SRV2 also disrupts yeast cell shape, resulting in abnormally large

mother cells (Figure 2B). Microscopic analysis of SRV2- or CAP1- transformed srv2!

cells showed normal sized mother cells. Moreover, Alexa-488-phalloidin staining of the

actin cytoskeleton showed that CAP1 rescues the actin organization defects of srv2!. In

addition, 69% of CAP1-transformed and 67% of SRV2-transformed srv2! cells displayed

robust actin cables (Graph Figure 2C).*

6*

F*

Figure 2. The cell growth and actin organization of CAP1 yeast.

A. Serial dilutions of srv2! yeast transformed with an empty vector, an SRV2 plasmid, and a

CAP1 plasmid, respectively, were grown in selective media, then plated on rich medium

(YPD) and grown at 25°C, 30°C, 35°C, and 37°C; B. DIC imaging of the transformed srv2!

cells. Cells were grown to log phase in selective medium at 25°C, and stained with Alexa488

phalloidin to visualize their actin cytoskeletons.

25°C!

pRS315!

SRV2!

CAP1!

pRS315!

SRV2!

CAP1!

30°C!

35°C! 37°C!

srv2"_!

srv2!_pRS315"

srv2!_SRV2"

srv2!_CAP1"

!"

#!"

$!"

%!"

&!"

'!!"

!"#$"%&'(")

*"'+&),-&.)/'01"+)

()*+'," *-.#" /01'"

CAP1 rescues the synthetic lethality of aip1! srv2! double mutant

As an additional in vivo test of the conservation between Srv2 and CAP1

functions, we tested the ability of the CAP1 plasmid to rescue the lethality of aip1!

srv2! double mutants. Both the aip1! and srv2! single mutants are viable, but crossing

them to each other results in lethality (or ‘synthetic lethality’). As expected, introduction

of an SRV2 plasmid will rescue these double mutants (Chaudhry et al., 2013). Similarly,

we observed that a CAP1 plasmid could rescue aip1! srv2! synthetic lethality at a range

of temperatures (Figure 3A), and support normal actin organization (Figure 3B).*

!!

*

*

*

*

*

*

6*

F*

Figure 3. CAP1 rescues the cell lethality and actin organization defects of aip1!

srv2! double mutants.

A. Serial dilutions of aip1!xsrv2! yeast transformed with an SRV2 plasmid, and a

CAP1 plasmid, respectively, were plated on rich medium (YPD) and grown at 25°C,

30°C, 34°C, and 37°C; B. Imaging of the transformed srv2! cells. Cells were grown

to log phase in rich medium (YPD) media at 25°C. Visualizing of their actin network

was obtained by staining with Alexa 488 phalloidin.

aip1! srv2!_SRV2"

aip1! srv2!_CAP1"

aip1! srv2!_"

CAP1"

CAP1"

25°C!

37°C!

35°C!

30°C!

SRV2"

SRV2"

CAP1 enhances cofilin mediated actin disassembly in vitro

From our in vivo data, we had a good indication that the actin regulatory functions

of Srv2 are conserved in CAP1. To better grasp the mechanisms by which CAP1

functions, we purified a number of proteins and performed biochemical assays

corresponding to Srv2 functions, namely disassembly and nucleotide exchange assays.

We performed these assays on full length CAP1 (here on referred to as CAP1), NCAP1

(the CC domain, and the HFD), and CCAP1 (the two proline rich regions, the WH2

domain, and the B-sheet domain) in the presence of cofilin, specifically human cofilin 1

(HsCof1).

First, we assessed the ability of CAP1 to enhance cofilin-mediated actin

disassembly. For this purpose, we performed disassembly assays on actin filaments, in

which 10% of the actin was fluorescently labeled with a pyrene dye. This dye has a

much higher fluorescence when actin is filamentous. At time zero in the assay, an agent

that binds actin monomers is added, so that there is no new assembly allowed, which

leads to a steady decay in the fluorescence signal as actin monomers dissociate from

filament ends over time. In the absence of cofilin, actin filaments disassemble slowly due

to the slow rate of subunit dissociation from filament ends (about 1 subunit per 3

seconds) (Figure 4). However, as expected, addition of HsCof1 to increase their rate of

disassembly (Figure 4). Similar to Srv2, CAP1 constructs alone (i.e. without cofilin) had

no effect on the rate of actin disassembly, but strongly enhanced the effects of HsCof1

(Figure 4). This was true for both CAP1 and NCAP1, whereas CCAP1 did not increase

HsCof1 activity (Figure 4). These observations suggest that NCAP1 enhances cofilin-

mediated actin disassembly, which is highly similar to the effects ofNSrv2.

CAP1 facilitates nucleotide exchange on actin monomers

Next, we explored the capacity of CAP1 to perform the second regulatory

function of Srv2, recharging actin monomers. To measure this, we performed nucleotide

exchange assays on actin monomers. This assay measures the increase in fluorescence of

labeled ATP as it binds to actin monomers. We performed nucleotide exchange assays

on actin monomers in the presence of the CAP1 constructs and HsCof1. Our results

show that actin will recharge itself relatively slowly, and that cofilin strongly inhibits this

rate of nucleotide exchange (Figure 5). However, there is a dramatic increase in the rate

of nucleotide exchange in the presence of CAP1 and CCAP1, even in the presence of

cofilin (Figure 5). NCAP1 does not increase the rate of nucleotide exchange (Figure 5).

This implies that the C-terminal half of CAP1 is responsible for this function.

Figure 4. The role of CAP1 in stimulating actin filament disassembly.

This measured the effects of full length CAP1, NCAP1, and CCAP1, in combination

with HsCof1, on the rate of actin disassembly. F-actin disassembly was induced by

vitamin D-binding protein in the presence of 2 µM F-actin (10% pyrene labeled), 100

nM CapZ, 250 nM HsCof1, and 750 nM of various CAP1 constructs.

.

!"

#!"

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&!"

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(!"

)!"

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+!"

#!!"

!" #!!" $!!" %!!" &!!" '!!" (!!" )!!" *!!" +!!"

flu

ore

scen

ce (

a.u

.)!

time (seconds)!

Actin!

HsCof1!

CAP1!

HsCof1+CAP1!

CCAP1!

HsCof1+CCAP1!

NCAP1!

HsCof1+NCAP1!

Contributions of CAP1 domains to CAP1 function

Previously, our lab has assigned Srv2 functions to 3 of its domains by mutating

conserved, surface-exposed residues; the HFD (Quintero-Monzon et al., 2009) enhances

cofilin-mediated severing of actin filaments, and the WH2 (Chaudhry et al., 2010) and "-

sheet domains (Mattilla et al., 2004, Chaudhry et al., 2013) that mediate nucleotide

exchange. Using site-directed mutagenesis, we created three corresponding mutations in

CAP1 (Figure 6A) and investigated their roles in the turnover of actin filaments.

The first mutant, CAP1-91, changes the RILKE motif in the HFD to AIAAA.

srv2-91 yeast are rounder, larger, and display a disorganized actin cytoskeleton

(Quintero-Monzon et al., 2009). The Srv2-91 protein is also unable to stimulate cofilin

Figure 5. The role of CAP1 in recharging actin monomers.

The rate of etheno-ATP exchange on 2 µM ADP-G-actin in the presence of 5 µM of

HsCof1 and 100nM of various CAP1 constructs was measured at 410nm emission

using a spectrophotometer.

0!

10!

20!

30!

40!

50!

60!

70!

80!

actin!

5 "M

HsC

of1!

100n

M C

AP1!

HsC

of1+

CA

P1!

100n

M N

CA

P1!

HsC

of1F

+NC

AP1!

100n

M C

CA

P1!

HsC

of1+

CC

AP1!

NE

ra

te (

A.U

) !

mediated actin disassembly in vitro (Quintero-Monzon et al., 2009). We explored

whether the CAP1-91 mutation similarly disrupts the actin disassembly function of

CAP1. To this end, we performed disassembly assays with NCAP1-91, comparing the

activity of this mutant to that of NCAP1. As shown above, NCAP1 stimulates actin

disassembly in the presence of cofilin. Our results illustrate that NCAP1-91 does not

increase the rate of actin disassembly in the presence of HsCof1 (Figure 6B). This

indicates that the CAP1-91 mutant disrupts residues integral to NCAP1 function in

promoting cofilin-mediated F-actin disassembly.

The other two mutants, CAP1-98, and CAP1-108, change residues in the C-

terminus of CAP1 to alanines. CAP1-98 exists in the WH2 domain and substitutes the

four conserved residues in the LKHV (LKKV in Srv2) motif into alanines. The

equivalent mutation in Srv2, Srv2-98, disrupts interactions of CSrv2 with G-actin,

abolishes the effects of CSrv2 of nucleotide exchange on actin, and causes defects in cell

growth and actin organization in vivo (Chaudhry et al., 2010). The CAP1-108 mutant

changes two proposed actin-binding residues in the "-sheet, an aspartic acid and a lysine,

into alanines. In yeast, the srv2-108 mutation causes partially depolarized actin

organization and impairs cell growth at higher temperatures (Mattilla et al., 2004), and

purified Srv2-108 protein fails to perform nucleotide exchange on actin monomers

(Chaudhry et al., 2013). We investigated the residues responsible for CCAP1 function by

performing nucleotide exchange assays using CAP1-98, and CAP1-108. Our findings

show that CAP1-98 is unable to perform nucleotide exchange, but that CAP1-108 does

mediate nucleotide exchange on actin monomers even in the presence of HsCof1 (Figure

6C).

We next looked at the genetic effects of CAP1-91 and CAP1-98. Serial dilutions

of srv2! cells transformed with CAP1-91 and CAP1-98 plasmids were grown at 25°C,

30°C, 34°C, and 37°C. Examination of cell growth indicated that neither CAP1-91

rescued temperature sensitive growth at 37°C (Figure 6D). The srv2! cells expressing

CAP1-91 and CAP1-98 also remained abnormally large and rounded, and displayed

defects in actin cytoskeleton (Figure 5E). Further, actin cables were present in only 35%

of CAP1-91 cells, and 30% of CAP1-98 cells (Graph Figure 6E).

P1! WH2! B-Sheet!P2!CC! HFD!

N-terminus! C-terminus!6*

F*

0!

5!

10!

15!

20!

25!

30!

35!

actin!

5 "M

HsC

of1!

100n

M C

AP1!

HsC

of1+

CA

P1!

100n

M C

CA

P1!

HsC

of1+

CC

AP1!

100n

M C

CA

P1

98!

HsC

of1+

CC

AP1

98!

100n

M C

CA

P1

108!

HsC

of1+

CC

AP1

108!

NE

rate

(a.u

.)!

!"!#

$!"!#

%!"!#

&!"!#

'!"!#

(!!"!#

!"!# (!!"!# $!!"!# )!!"!# %!!"!# *!!"!# &!!"!# +!!"!# '!!"!# ,!!"!#

flu

ore

scen

ce (

a.u

.)!

time (seconds)!

NCAP1!

HsCof1+NCAP1!

NCAP1-91!

HsCof1+NCAP1-91!

HsCof1+NCAP1-P1!

actin!

HsCof1!

E*

Figure 6. The biochemical and genetic properties of CAP1 mutants.

A. The structure of CAP1 indicating the locations of the mutations; B. The effects of

CAP1-91, in conjunction with HsCof1, in actin disassembly assays. F-actin

disassembly was induced by vitamin D-binding protein in the presence of 2 µM F-

actin (10% pyrene labeled), 100 nM CapZ, 250 nM HsCof1 and 750 nM of NCAP1-

91; C. The effects of the 100nM of CAP1-98 and CAP1-108 constructs on the rate of

etheno-ATP exchange on 2 µM ADP-G-actin in the presence of 5 µM of HsCof1. The

nucleotide exchange abilities of the mutant CAP1 constructs were measured at 410nm

emission using a spetrophotometer; D. Serial dilutions of CAP1 mutants were plated

on YPD and grown at 25°C, 30°C, 34°C, and 37°C; E. DCI imaging of Srv2! cells

transformed with CAP1-91, or CAP1-98. Cells were grown to log phase in drop-out

media, fixed, and stained with Alexa488 phalloidin in order to visualize the actin

network.

@*

G*

CAP1-98!

CAP1!

CAP1-91!

CAP1!

CAP1-91!

CAP1-98!

25°C!

34°C!

30°C!

37°C!

pRS315!

srv2"_!

pRS315!

srv2!_CAP1"

srv2!_CAP1-91"

srv2!_CAP1-98"

!"

#!"

$!"

%!"

&!"

'!!"!"#$"%&'(")

*"'+&),-&.)/'01"+)

()*+'," -./'" -./'01'" -./'01&"

srv2!_pRS315"

Discussion

Srv2/CAP1 is a conserved protein in yeast, mammals, and many other

species. Most research has been performed on the yeast homologue, Srv2. We

used genetic and biochemical approaches to show that the regulatory roles of yeast

Srv2, specifically stimulating cofilin mediated actin disassembly and promoting

nucleotide exchange on actin monomers, are conserved in mammalian CAP1. We

demonstrate that CAP1 is capable of rescuing an srv2! phenotype, and has

biochemical functions indistinguishably from those of Srv2 in promoting cofilin

mediated actin filament disassembly and catalyzing nucleotide exchange on actin

monomers.

We also used mutations and truncations to assign the activities of mouse

CAP1 to specific domains and sequences, which revealed a tight correlation with

yeast Srv2. NCAP1 was responsible for the effects on actin filament disassembly,

as has been shown for NSrv2, and the CAP1-91 mutant abolished this activity in

the same way the equivalent mutation (Srv2-91) does in yeast Srv2 (Quintero-

Monzon et al., 2009, Chaudhry et al., 2013). These parallels were also observed

in vivo. The srv2-91 yeast cells are abnormally large, rounded, and have a

depolarized actin network (Quintero-Monzon et al., 2009) Likewise, a wild type

CAP1, but not mutant CAP1-91 plasmid will rescue srv2! phenotypes. This

suggested that the specific site mutated in the ’91’ mutation (located in the N-

terminal HFD domain) mediates the same conserved function in yeast and mouse

Srv2/CAP homologues.

Similar to the ability of yeast CSrv2 to catalyze nucleotide exchange on

actin monomers (Mattilla et al., 2004, Chaudhry et al., 2010, Chaudhry et al.,

2013), we found that CCAP1 has this activity. We further demonstrated that

CCAP1 is able to perform this function even in the presence of cofilin (which is

normally inhibitory to nucleotide exchange). Thus, not only is CCAP1 able to

facilitate nucleotide exchange on actin monomers, but it also relieves the

inhibitory effects of cofilin on nucleotide exchange.

As with NCAP1, we also sought to further elucidate the locations involved

in CCAP1 function. To this end, we generated two mutations, CAP1-98 in the

WH2 domain, and CAP1-108 in the "-sheet, that correspond to loss of function

mutations in Srv2. Srv2-98 is unable to promote nucleotide exchange on actin

monomers and mutant srv2-98 yeast show defects in cell morphology, actin

organization, and growth at higher temperatures (Chaudhry et al., 2010).

Similarly, a mutant mouse CAP1-98 plasmid was unable to rescue the defects of

srv2! yeast, and biochemically, CAP1-98 failed to promote nucleotide exchange

on actin monomers in the presence or absence of cofilin. Analogous to the results

of CAP1-91, this result shows the WH2 domain’s function in nucleotide exchange,

and localizes the residues responsible. Furthermore, it shows that the function and

location of the active site in this WH2 domain is highly conserved from yeast Srv2

to mammalian CAP1.

The third mutant, CAP1-108, was also assessed for its CCAP1 function.

Srv2-108 is unable to recharge actin monomers (Chaudhry et al., 2013) and Srv2-

108 yeast are temperature sensitive and have a perturbed actin network (Mattilla et

al., 2004). Somewhat surprisingly to us, CAP1-108 was still able to promote

nucleotide exchange on actin monomers, even in the presence of cofilin. This

means either that the residues responsible for the "-sheet’s contribution to

nucleotide exchange are not the same in mammals as they are in yeast, or that the

"-sheet does not contribute to nucleotide exchange as much, or at all in CAP1.

However, the fact that malaria CAP, which consists only of a "-sheet (no other

domains), is capable of performing nucleotide exchange on actin monomers

(Makkonen et al., 2013) makes the latter scenario seem unlikely. To further

investigate this result, we are now combining CAP1-108 with another mutation in

the same domain, predicted to contact G-actin (Mattilla et al., 2004), in an attempt

to generate a double mutant with more impaired binding to G-actin.

Our results establish that the functions of yeast Srv2 to accelerate actin

cytoskeleton turnover, namely enhancing cofilin mediated F-actin severing,

and catalyzing nucleotide exchange on actin monomers, are remarkably

well conserved in mammalian CAP1. Furthermore, we demonstrate that

the locations of these functions, the HFD and WH2 domains, respectively,

are conserved from yeast to mammals. However, further investigations

need to be made into the function of the "-sheet domain of CAP1. We have

also gathered results showing that NCAP1 oligomerizes, but we do not

know if it, like NSrv2, forms a hexamer (Chaudhry et al., 2013). Also,

whether the actin regulatory functions of Srv2 are conserved in the other

mammalian homologue, CAP2 has yet to be determined.

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