This document summarizes an honors thesis project investigating the localization of ESCRT protein complexes during cytokinesis in C. elegans embryos. The author used fluorescent tagging and immunofluorescence to show that: 1) ESCRT-I co-localizes with the midbody marker ZEN-4 during cytokinesis. 2) ESCRT-I and ESCRT-III show fixed co-localization throughout cytokinesis, though this is less distinct in later embryonic cell stages. 3) Both ESCRT-I and ESCRT-III localize in proximity to the midbody marked by ZEN-4. 4) Preliminary work suggests ESCRT-I more directly

1. 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

2. 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

3. 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.

4. 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

5. 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

6. 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

7. 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-32Merge
A
Merge GFP ESCRT-I Ab VPS-32
B
GFP ESCRT-I Ab VSP-32Merge
C

8. 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-32Merge
mCherry ZEN-4

9. 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.

10. 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

11. 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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