Emily  Baumann  Senior  Year  Honors  in  Research  Thesis  Spring  2015   INTRODUCTION Cytokinesis is the process of physically separating a cell into two daughter cells, which

maintains the existence of multicellular organisms. During mammalian cell cytokinesis, the

plasma membrane is constricted to form an intracellular bridge that is eventually severed to form

two daughter cells (Straight and Field, 2000; Alsop and Zhang, 2003). Cytokinesis is divided

into two steps, orchestrated by many protein complexes. The first step involves septin complex-

driven contractile ring constriction between daughter cells, which divides the cytoplasm. The

second step involves the abscission of daughter cells. Abscission has been suggested to be

orchestrated by both the septin complex and the endosomal sorting complex required for

transport (ESCRT) machinery. Abscission involves the narrowing of the intracellular bridge

between daughter cells, followed by membrane fission (Green et al., 2012; Fededa and Gerlich,

2012). The midbody complex is made up of antiparallel microtubules and is located in the center

of the intracellular bridge. The midbody serves as a scaffold for the recruitment of the protein

complexes involved in abscission (Green et al., 2013; Guizetti and Schermelleh, 2011; Schiel

and Prekeris, 2013). In this project, the midbody will be fluorescently labeled and serve as a

marker for examining protein complex distributions during abscission.

From archaea to eukaryotes, ESCRT mediated membrane scission is conserved. In most

organisms, protein components of the ESCRT machinery assemble on the cytoplasmic surface of

the intracellular bridge to facilitate membrane remodeling and formation of two daughter cells.

The ESCRT machinery is composed of five protein complexes: ESCRT-0, -I, -II, -III, and -IV.

Of these, three are thought to play significant roles in cytokinesis: ESCRT-I, -III, and -IV (Dukes

et al., 2008; Elia et al., 2011; Morita et al., 2010). Previous research in mammalian cells suggests

that these complexes are recruited sequentially to the intracellular bridge (Elia et al., 2011), and

ESCRT-III is directly required for the final membrane scission step between daughter cells

(Bajorek et al., 2009). Spatial and temporal correlation of the ESCRT-III component CHMP4B

(called VPS-32 in worms) with the site of abscission further suggests a direct role for ESCRT

machinery in membrane scission (Elia et al., 2011). The exact mechanism that facilitates the

recruitment and stable distribution of ESCRT components at the midbody is unknown. In

addition, the exact temporal recruitment order of the ESCRT machineries is yet to be determined.

Septin components also function during cytokinesis and localize to the cleavage furrow

between dividing cells where they become components of the actin-myosin contractile ring

structure (Glotzer, 2001). The contractile ring is the driving force behind proper cytoplasm

division during cytokinesis. Within the C. elegans genome, the UNC-59 and UNC-61 loci encode

the only two septins and both are required for normal contractile ring formation (John et. al

2007). In mammalian cells grown on plastic, abscission fails in the absence of septin function

(Field et al., 1996; Lukoyanova et al., 2008).

Completion of cytokinesis can take as long as three hours after the initial formation of an

intracellular bridge in mammalian cells. However, the association between ESCRT-I and

membranes is transient and weak. This weak attraction suggests that other proteins are present to

assist the prolonged localization of ESCRT-I to the midbody, ensuring proper abscission. In

mammals, the ESCRT-I subunit Tsg101 is directed to the midbody by the microtubule bundling

protein Cep55 (Agromayor and Martin-Serrano, 2013). Previous research using C. elegans

shows that ESCRT-I localizes to the midbody during embryogenesis, even though worms lack a

Cep55 homolog. This data suggests that an alternative mechanism enables ESCRT components

to be involved in cytokinesis (Green et al., 2013), which may also function in cells outside of

nematodes. Our research will investigate the possible role of the septin complex in regulating

ESCRT localization to the midbody, using the C. elegans early embryo as a model system.

In summary, previous work suggests that both ESCRT and septin protein complexes are

involved in cytokinesis, but whether their individual functions are dependent on one another is

unknown. I hypothesize that the septin and ESCRT complexes function together to facilitate

membrane scission during cytokinesis. In addition, I hypothesize that there exists a strong co-

localization tendency of various ESCRT machineries to the midbody. These hypotheses will be

tested using the specific methods detailed below.

METHODS

Confocal Microscopy Analysis: Endogenous Localization of ESCRT-I with the Midbody

Fluorescence microscopy was used to image embryos from transgenic worms to

determine the localization of ESCRT protein components to the midbody during cytokinesis. To

examine the localization of ESCRT-I, I created transgenic C. elegans stably expressing mCherry-

tagged ZEN-4 (a midbody marker) together with GFP-tagged ESCRT-I. The Audhya lab had

already generated worm lines expressing the fluorescently tagged proteins individually. Worm

lines expressing only one fluorescent tag were also used for some assays. Specifically, I

conducted genetic crosses and isolated homozygous strains that expressed two fluorescently

labeled markers. I confirmed the stable expression of two fluorescently tagged protein

components using my lab’s Nikon Confocal Microscope. For imaging experiments, C. elegans

were dissected and embryos were immobilized on a glass slide. Localization of ESCRT protein

components was viewed using a confocal microscopy.

Immunofluorescence Assays

During my research within the Audhya lab I investigated the temporal distribution of

multiple ESCRT protein components during cytokinesis. I used C. elegans with fluorescently

labeled ESCRT-I and stained them with a fluorescently labeled antibody against ESCRT-III. I

choose C. elegans that were very young adults, having few embryos, along with adults having

approximately 10 embryos. By using C. elegans from multiple stages of adult life I was able to

capture the ESCRT-I/-III localizations within embryos of different cell division stages. I viewed

the localization of both ESCRT components using confocal microscopy.

In parallel with these studies, I examined the distributions of endogenous ESCRT-I,

ESCRT-III, and septin complexes using immunofluorescence. My lab previously generated

antibodies against ESCRT-I (TSG-101) and ESCRT-III (VPS-32). C. elegans with fluorescently

labeled midbody marker ZEN-4 (mCherry) will be stained with fluorescently labeled antibodies

directed against either ESCRT-I or ESCRT-III (both Cy5-labeled). Using confocal microscopy,

the distributions of the proteins in reference to the midbody marker ZEN-4 was observed.

I also used immunofluorescence to study the septin and ESCRT complex localization

dependencies. I observed ESCRT complex localization in C. elegans that fail to express one

septin. In contrast to the essential role of the septins in human tissue culture cells, mutant C.

elegans animals that lack the septins are viable. To observe how the abscission-dependent

ESCRT-III, and ESCRT-I protein components localize during cytokinesis within septin deficient

C. elegans embryos, I also used fluorescence confocal microscopy. I specifically observed the

localization of ESCRT-III and ESCRT-I to the plasma membrane within embryos undergoing

cytokinesis. A particular worm line, ∆UNC-61, was used which expressed a mCherry

fluorescently tagged plasma membrane marker. In preliminary work, I have determined succinct

methods to generate worm lines that lack the septins. Embryos were stained with a fluorescently

labeled antibody directed against either ESCRT-III or EXCRT-I (both Cy5-labeled).

Preliminary Trials of Microinjection Experiments  

In addition, to further determine the localization dependencies between the septin and

ESCRT complexes, I spent majority of summer 2014 acquiring an RNA interference (RNAi)

skillset. The development of this skillset was necessary for proper RNAi assay completion. My

goal at the beginning of this project was to deplete C. elegans of endogenous ESCRT complex

components by RNAi and examine if septin components are mislocalized during cytokinesis.

The RNAi assay is completed using a microinjection apparatus. My laboratory currently uses a

Narishige IM-30 Motor Drive Microinjection system. The apparatus uses hand-pulled glass

needles made from glass capillary tubes. I experimented with multiple needle pulling apparatuses

in order to obtain the correct sized injection tip.

I completed preliminary trials of my own microinjection experiments, which helped me

gauge my understanding of the RNAi process. I used a simple solution containing a fluorescently

tagged marker. By injecting this into multiple trials of wild type C. elegans, and using confocal

microscopy, I was able to determine if the solution was injected correctly. Success RNAi through

microinjection depends on the injection of C. elegans in the gonads. If the gonads are not

injected, the RNAi will not act downstream to block gene expression.

RESULTS

Endogenous Localization of ESCRT-I with the Midbody

I used fluorescence confocal microscopy to image embryos from a C. elegans worm line,

stably expressing mCherry-tagged ZEN-4 (a midbody marker) together with GFP-tagged

ESCRT-I. Using this worm line allowed me to investigate the localization of the ESCRT-I

protein component during cytokinesis. I imaged multiple embryos in channels 488nm, for GFP-

tagged ESCRT-I, and 561nm for mCherry-tagged ZEN-4. The confocal microscope software

then merged both channels to create a single image. The merge depicted co-localization of

ESCRT-I and ZEN-4 by the presence of yellow puncta (color from overlay of green and red) as

shown in Figure 1.

Figure 1. C. elegans worm line stably expressing fluorescently tagged ZEN-4 (mCherry) and ESCRT-I (GFP). Confocal microscopy imaging reveals the co-localization of ZEN-4 (midbody marker) with ESCRT-I protein component in merge (left, yellow markers). Individual channels show expression of ZEN-4 (right, red) and ESCRT-I (middle, green). Temporal Distribution of Multiple ESCRT Protein Components

To further my understanding of how the ESCRT protein components localize during

cytokinesis, I used immunofluorescence to visualize the ESCRT-I/-III proteins in C. elegans

embryos. I choose C. elegans adults, with fluorescently labeled ESCRT-I, having varying

numbers of embryos (approximately between 4-10). This careful selection maximized my

chances of observing the localization between ESCRT-I and ESCRT-III in multiple embryonic

cell stages (2 cell stage to 6 cell stage). After staining C. elegans with fluorescently labeled

antibody against ESCRT-III, I used multiple channels (488nm and 638nm) of a confocal

microscope to visualize ESCRT component co-localizations. Overall, whether the embryo is in

an early-phase or late-phase of embryonic development, there is a fixed localization between

ESCRT-I and ESCRT-III. Figure 2 depicts early (A-B, 2-4 cell stage) and late cell stage (C,

multi-cell) embryos. Early-phase embryos appear to have slightly more fixed co-localizations

between ESCRT-I and ESCRT-III, when compared to late-phase embryos. Early cell divisions

do show a dedicated co-localization, where ESCRT-I and ESCRT-III are markedly in the same

area. As the embryo matures beyond the 4-cell stage, there are more ESCRT components than an

early stage embryo. Therefore, observing the co-localization between individual ESCRT

components becomes less obvious.

Figure 2. C. elegans embryos stained with antibody against ESCRT-III, with stable expression of GFP-tagged ESCRT-I. Worm line expressing GFP-tagged ESCRT-I stained with antibody against ESCRT-III shows co-localization of protein components (merge, left). Individual channels show expression of ESCRT-I (middle, green) and ESCRT-III (right, grey).

GFP ESCRT-I Ab VPS-32 Merge

A

Merge GFP ESCRT-I Ab VPS-32

B

GFP ESCRT-I Ab VSP-32 Merge

C

Confocal microscopy images of a two-cell stage embryo (A), four-cell stage embryo (B), and multi-cell stage embryo (C). Distribution of the ESCRT-I/-III Protein Components in reference to the Midbody

Using confocal microscopy, the distributions of the ESCRT-I/-III proteins in reference to

the midbody marker ZEN-4 was observed. Using a C. elegans worm line stably expressing a

fluorescent (mCheery) midbody maker for ZEN-4, I stained embryos with antibodies directed

against ESCRT-I and ECRT-III. Imaging the embryos in channels 561nm and 638nm, I was able

to see each ESCRT component’s location in relation to the midbody, as seen in Figures 3-4.

Figure 3. C. elegans embryo stained with antibody against ESCRT-I, with stable expression of ZEN-4 (midbody marker). Worm line expressing mCherry-tagged (red) ZEN-4 midbody marker stained with antibody against ESCRT-I shows co-localization of protein components (merge, left). Individual channels show expression of ESCRT-I (right, green) and midbody marker ZEN-4 (middle, red).

Figure 4. C. elegans embryo stained with antibody against ESCRT-III, with stable expression of ZEN-4 (midbody marker). Worm line expressing mCherry-tagged (red) ZEN-4 midbody marker stained with antibody against ESCRT-III shows co-localization of protein components (merge, left). Individual channels show expression of ESCRT-III (right, grey) and midbody marker ZEN-4 (middle, red).

Merge Ab TSG-101

mCherry ZEN-4 Ab VPS-32 Merge

mCherry ZEN-4

ESCRT Complex Localization in C. elegans that Fail to Express Septins

By using immunofluorescence I was able to visualize localization dependencies between

the septin and ESCRT complexes. By staining ∆UNC-61 C. elegans with antibodies against

ESCRT-I or ESCRT-III, I saw the location of the ESCRT components in relation to the plasma

membrane. When completing multiple trials of this immunofluorescence assay, I noticed that the

plasma membrane didn’t fluorescence strongly when viewed in the 561nm channel of a confocal

microscope. Further investigation revealed that the methanol wash was depleting the color of the

plasma membrane. Figure 5 depicts the most representative images of the ∆UNC-61 embryos

stained with antibodies against either ESCRT-I or ESCRT-III. ESCRT-I appeared to be more

directly localized to the plasma membrane, in relation to ESCRT-III. Further experiments are

needed to fully comprehend the localization of ESCRT-I/-III to the plasma membrane in C.

elegans deficient of one septin.

Figure 5. ∆UNC-61 C. elegans embryos stably expressing mCherry tagged plasma membrane marker stained with TSG-101 and VPS-32 antibodies. ∆UNC-61 worm line was used which expressed a mCherry fluorescently tagged plasma membrane marker. Embryos were stained with an antibody against either ESCRT-I (right) or ESCRT-III (left). Images show the protein component localization to the plasma membrane.

Preliminary Trials of Microinjection Experiments

Learning how to operate my laboratory’s microinjection apparatus was very intuitive and

gave me the opportunity to teach other laboratory personnel. Overall, my efforts furthered my

laboratory’s understanding of the microinjection process and increased the success rate of

obtaining injected C. elegans. I did successfully inject multiple trials of wild type C. elegans

with a simple solution containing a fluorescently tagged marker. I could apply my microinjection

practice to injecting worms with RNAi. Specifically, I would like to use my RNAi injection

techniques to deplete either ESCRT-I or ESCRT-III complex proteins within C. elegans strains

expressing fluorescently labeled septins. Live worm imaging (using strains expressing GFP-

tagged septins) and immunofluorescence techniques (using antibodies available in the lab) would

then be used to demonstrate whether septin components still localize normally to the midbody in

the absence of ESCRT complex components.

SUMMARY

Since 2012, I have been an undergraduate researcher within the Audhya lab using C.

elegans as a model organism. For my most recent project I read multiple articles that suggested

the ESCRT and septin protein complexes are involved in cytokinesis. However, research wasn’t

clear of whether each complex’s individual functions were dependent on one another. I

hypothesized that the septin and ESCRT complexes function together to facilitate membrane

scission during cytokinesis. My experiments showed that there is a localization of the ESCRT

protein components to the site of abscission, in the absence of one septin. This may suggest that

the ESCRT and septin complexes are not dependent on one another for localization to the site of

abscission. However, additional experiments are needed to fully investigate if the complexes

function synergistically.

In addition, I hypothesized that there is a strong co-localization tendency of various

ESCRT machineries to the midbody. Throughout completing various immunofluorescence

assays, I did see a strong co-localization between ESCRT-I/-III and midbody marker ZEN-4.

Overall, my research does add to the growing repertoire of information about the protein

components involved in the abscission phase of cytokinesis. Further experimentation is needed

to investigate the functionality of the ESCRT and septin components, and fully decipher their

spatial distribution during abscission. My time spent in the Audhya lab has taught me more than

just laboratory techniques; my experiences gave me a thorough understanding of formulating a

scientific hypothesis, designing experiments, analyzing results, and formal scientific writing. I

was also given the opportunity to “co-mentor” several undergraduate students within the Audhya

lab. This experience has advanced my teaching skills and communication abilities.

I would like to give thanks to Dr. Anjon Audhya and all Audhya lab graduate students

and staff for their help and guidance throughout my research.

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