2014 Front Range Cytoskeleton Meeting Program
May 29, 2015
University of Colorado, Boulder
Boulder, CO
1 THANK YOU FOR YOUR SUPPORT!
This meeting was funded by the following generous
sources.
Molecular, Cellular, and Developmental Biology Department
University of Colorado, Boulder
Cytoskeleton, Inc. – The Protein Experts
Nikon Corporation
Biology Open
2 Meeting Location & Directions
Parking: FRCM will provide parking in Lot 436 (see map). There will be an attendant at the
lot from 7:00 am until 9:00 am. Tell the attendant that you are with the meeting and they will
give you a pass for your car. If you arrive after 9:00 am, park in 436, come to the meeting and
we will work it out.
Lot 436 is easy to access for those coming from out-of-town. From either Foothills Parkway or
US 36 (28th St in Boulder), head west on Colorado Blvd. Make a left at the first traffic light
west of US 36/28th St. onto Regent Drive (be in the far left lane of this double left). The first
left off of Regent Drive is the entrance to lot 436.
The meeting is in Gold Biosciences. From Lot 436, walk west on Colorado Blvd past the
intersection with Folsom St. and the stadium on your right. Gold Bioscience is the first building
past the stadium. Gold faces the plaza with the buffalo sculpture. Enter the breezeway in
Gold Biosciences and then enter the doors on the right with stairs down to the meeting checkin and lecture hall (Gold A2B70).
The talks will be in Gold A2B70.
Lunch and the poster session will be in Gold A120 (Wood Classroom).
3 Post-meeting dinner / drinks location
Dinner Event: We will have "heavy appetizers" and drinks at the Koenig Alumni Center on the
Boulder campus. This nice venue is a short walk from Gold Biosciences. We are requesting a
$10 per person contribution for this event - payable at the door. You will need your license to
be admitted because alcohol is being served. Registrants will receive a separate invitation to
this event - please RSVP so we can get an accurate head count. Directions to Koenig will be
distributed at the meeting.
4 Nick Galati Keynote Speaker:
Karen Oegema (UC San Diego)
A Chemical Biology Approach to Centriole Function in Mammals
Centrioles are microtubule-based organelles that direct the formation of centrosomes and cilia.
Supernumerary centrosomes are linked to aneuploidy and are a common feature of human cancers.
Despite their broad impact on cell physiology, centriole function has been challenging to study in
mammalian cells due to the lack of a facile methodology for specifically, persistently, and reversibly
removing this organelle from cells. To facilitate analysis of centrioles, we developed centrinone – a
specific small molecule inhibitor of PLK4, the kinase that initiates centriole assembly. Treatment with
centrinone at sub-micromolar concentrations allows persistent and reversible depletion of centrioles
from cultured human and mouse cells, leading to loss of centrosomes and primary cilia. To test the
importance of centrosomes in the proliferation of normal and cancerous human cells, we developed
centrinone, a specific reversible inhibitor of Plk4—the kinase that initiates centriole assembly.
Centrinone enables routine depletion of centrosomes from human and other vertebrate cells.
Surprisingly, centrosome loss irreversibly arrested normal cells in a senescence-like G1 state via a p53dependent mechanism that was independent of DNA damage/stress/Hippo signaling, extended mitotic
duration, or segregation errors. In contrast, cancer cell lines with normal or amplified centrosome
numbers could proliferate indefinitely following centrosome loss, indicating that cells with cancerassociated mutations fundamentally differ from normal cells in their response to centrosome loss. In
transformed cell lines with different intrinsic levels of centrosome amplification prior to centriole
depletion, centrinone washout triggered a wave of de novo assembly and initial overduplication,
followed by recovery to the level of amplification observed prior to centriole removal. Analysis of this
recovery process indicated that overduplication is intrinsically balanced by removal of cells with extra
centrioles through multipolar mitosis and death. These results suggest that centriole number set points
in cancer cell lines result from a dynamic equilibrium between centriole overduplication and removal of
cells with extra centrioles rather than being determined by historical overduplication events.
5 Meeting Schedule
TIME
Event
TITLE
8:00-8:50a
Check In / Registration (Location: Gold A2B70)
8:50-9:00a
Opening Remarks – Mark Winey (Location: Gold A2B70)
9:00-9:20a
Rita Miller (OSU)
SUMO interacts with multiple classes of MAPs
9:20-9:40a
Anthony Mangan (CU – Anschutz)
Directing Traffic: The Role of Microtubules and Actin
Dynamics in the Establishment of an Apical Lumen
9:40-10:00a
Todd Blankenship (Univ of
Denver)
Planar polarized Rab35 functions in an oscillatory
system to drive interface contraction
10:00-10:20a
Colby Fees (CU – Anschutz)
Chromosome segregation depends on a negativelycharged region of the β-tubulin carboxy-terminal tail
10:20-10:40a
Coffee Break
10:40-11:00a
Jennifer DeLuca (CSU)
TBA
11:00-11:20a
Lydia Heasley (CU – Anschutz)
TBA
11:20-11:40a
Andy Hoenger (CU – Boulder)
Structure, function and microtubule interaction patterns
of hetero-dimeric kinesins
11:40-12:00p
Dinah Loerke (Univ of Denver)
Biomechanics of cell intercalation
12:00-1:00p
Lunch – Location: Gold A120 (Wood Classroom)
**Vegetarian and gluten free lunch options will be available. However, folks with strict diets may
consider bringing a sack lunch from home.
1:00-2:30p
Poster Session (Location: Gold A120)
2:30-3:30p
Keynote: Karen Oegema (UCSD)
3:30-3:40p
FRCM Business
3:40-4:00p
Coffee Break
4:00-4:20p
Diego Krapf (CSU)
A Chemical Biology Approach to Centriole Function in
Mammals
TBA
6 4:20-4:40p
Meredith Betterton (CU – Boulder)
Kinetochore movements and mitotic spindle dynamics
in fission yeast kinesin-8 mutants
4:40-5:00p
Jim Bamburg (CSU)
A prion-dependent signaling pathway in neuronal
oxidative stress and neurodegenerative disorders.
5:00-5:10p
Concluding Remarks and Business (Todd Blankenship)
5:15-
Drinks and Dinner at the Koenig Alumni Center
7 Talk Abstracts:
9:00-9:20a
Rita Miller (OSU)
SUMO interacts with multiple classes of MAPs
Stu2 is the yeast homologue of XMAP215 and promotes microtubule dynamics by facilitating
the loading of tubulin dimers onto microtubule ends. We previously identified four different
classes of microtubule-associated proteins that interact with SUMO: Kar9, Bim1/EB1,
Bik1/CLIP170, and Pac1/LIS. Here we show that a fifth class of MAP, Stu2/XMAP215,
interacts with the Small Ubiquitin-like Modifier (SUMO). Sumoylation is a post-translational
modification that covalently attaches the SUMO protein to target substrates. Whereas
sumoylation regulates many cellular processes such as cellular transport, protein stability and
transcription, it has only recently been shown to regulate spindle positioning. We have
previously shown that the LIS1 homologue Pac1 is modified in vivo by SUMO and ubiquitin.
Pac1 regulates dynein activity and is important for recruiting dynein to the plus end of the
microtubule. Dynein is subsequently “off-loaded” to the cortex where it pulls on cytoplasmic
microtubules to move the mitotic spindle across the bud neck, a key step in positioning the
mitotic spindle. Although Pac1 plays a vital role in microtubule function, little is known about
how it is regulated. Here we use domain mapping to show that the alpha-helical region of Pac1
is important for its interaction with Smt3 and Ubc9. We also show that two point mutations
within the alpha-helical domain, K15R/A and K20R, disrupt Pac1 interaction with Smt3/SUMO
and Ubc9. We also show that PAC1 interacts with STU2. The C-terminus of Stu2 is sufficient
for this interaction. Using two-hybrid analysis, we show that Stu2 interacts with Smt3/SUMO
and other key members of the sumoylation system. Using an in vitro sumoylation assay, four
shifted bands of Stu2p can be observed. Inactivation of the Ulp1p SUMO protease with a
temperature sensitive mutant results in Stu2 displaying a higher molecular weight band in vivo.
These results indicate that Stu2 can be conjugated by SUMO. In whole cell extracts, Pac1
shifts more extensively than Stu2 by western blotting. We suggest that sumoylation may be a
general mechanism for regulating microtubule-associated proteins.
8 9:20-9:40a
Anthony Mangan (CU – Anschutz)
Directing Traffic: The Role of Microtubules and Actin Dynamics in the Establishment of
an Apical Lumen
Epithelial cells are structurally and functionally polarized to transport specific molecules while
maintaining a trans-epithelial barrier. Additionally, epithelial cells coordinate their polarization
with neighboring cells to form an apical lumen, a key step in the establishment of epithelial
tissue architecture, and thereby function. We have shown that midbody formation during
telophase is the first symmetry-breaking event that determines the site of apical lumen
formation between two epithelial cells. Furthermore, studies in our lab have shown that tight
junction protein Cingulin is recruited to the midbody during cell division, and that this
recruitment plays a key role in marking the site of lumen formation. Despite recent advances in
our understanding of the mechanisms mediating apical lumen formation, many questions
remain unanswered. For example, the machinery mediating Cingulin recruitment to the
midbody during apical lumen formation remain essentially unknown.
In this study we focus on identifying the machinery mediating Cingulin recruitment to the
midbody during late telophase. We demonstrate that both microtubule binding and actin
networks are required for establishing the site of the apical lumen. First, we have shown that
Cingulin binds to microtubule C-terminal tails (CTTs) and that this binding is likely regulated by
glutamylation. Second, we completed immunoprecipitation, immunofluorescence, and
proteomics analysis of synchronized epithelial cells in telophase and identified components of
the WAVE/SCAR complex as putative regulators of Cingulin recruitment to the midbody. Since
Rac1 is known to activate the WAVE/SCAR complex, we next demonstrated that Rac1 is also
present at the midbody and that Rac1 activation is required for Cingulin recruitment to the
midbody during apical lumen formation. Finally, we observed the formation of “actin flares” at
the midbody during late telophase and that these “actin flares” may initiate cell polarization and
apical lumen formation during epithelia morphogenesis. This data supports a combinatorial role
of microtubules and actin in the coordination and regulation of the apical membrane initiation
site and forming lumen.
9 9:40-10:00a
Todd Blankenship (Univ of Denver)
Planar polarized Rab35 functions in an oscillatory system to drive interface contraction
The control of cell shape is a fundamental property required for epithelial tissue architecture
and function. Here, we show that a Rab protein, Rab35, is planar polarized during epithelial
tissue remodeling. We use CRISPR-mediated knock-in to examine the dynamics of an
endogenously-tagged Rab protein. Rab35 compartments are more numerous and dynamic at
contractile interfaces of actively intercalating cells. Individual compartmental behaviors have
lifetimes of ~120 seconds, and correlate with periods of rapid interface contraction. Although
tensile actomyosin forces have been conventionally thought to drive interface contraction,
initiation of Rab35 compartmental behaviors does not require Myosin II function. However,
when Myosin II function is disrupted, Rab35 compartments do not terminate and continue to
grow into large elongated, tubular structures. These compartments are contiguous with the cell
surface, and are likely hubs of endocytosis. Rab35 activity is controlled by the AnteriorPosterior patterning system, and Rab35 function is required for progressive interface
contraction. Finally, we demonstrate that Rab35 is likely involved in a common contractile cellshaping mechanism, as cells undergoing apical constriction during mesoderm invagination
also form Rab35 compartments at their shrinking surfaces, but with distinct kinetics and an
absence of planar polarity. Our results suggest that the coordination of membrane trafficking
and cytoskeletal forces converge on Rab35 compartmental behaviors to direct cell shaping
events.
10 10:00-10:20a
Colby Fees (CU – Anschutz)
Chromosome segregation depends on a negatively-charged region of the β-tubulin
carboxy-terminal tail
Proper chromosome segregation requires carefully choreographed interactions between
kinetochores and dynamic spindle microtubules. Whereas the roles of kinetochore proteins are
relatively well understood, how tubulin proteins contribute to the fidelity of chromosome
segregation is poorly understood. Here we investigate the negatively-charged carboxy-terminal
tail (CTT) domains of the α- and β-tubulins, which are thought to promote electrostatic
interactions with kinetochore proteins. CTT sequences are highly variable across species and
tubulin isotypes, and are major sites of post-translational modifications. CTTs are, therefore, a
possible point of regulating kinetochore-microtubule interactions that determine the fidelity of
chromosome segregation. Using a series of mutants that alter or ablate CTTs of α- and βtubulin in budding yeast, we identify a specific role for β-CTT in chromosome segregation.
Mutant strains lacking the β-CTT exhibit delayed progression into anaphase and elevated rates
of chromosome loss. In contrast, mutants lacking the β-CTT appear similar to wild type. Using
live cell imaging to measure the dynamics of kinetochores labeled with Nuf2-GFP, centromeres
labeled with CENP-A/Cse4 –GFP, and single centromeres labeled with CENIV-GFP, we show
that loss of the β-CTT disrupts the bi-orientation of sister kinetochores. To elucidate the
molecular role of the β-CTT, we map the residues that are necessary for function, and identify
a short region of negatively-charged residues. Altering the charge of these residues disrupts
chromosome segregation and microtubule dynamics. Furthermore, this negatively charged
region may play an important role in facilitating interactions between the CTT and microtubule
binding proteins. We provide evidence that this region supports the activity of the kinetochore
protein, Ndc80. Based on these results, we propose that the β-CTT promotes proper
chromosome segregation in two ways; by regulating the dynamics of spindle microtubules and
by tuning kinetochore-microtubule interactions.
11 10:40-11:00a
Jennifer DeLuca (CSU)
One of the most important regulatory aspects of chromosome segregation is the ability of
kinetochores to precisely control their attachment strength to microtubules. Central to this
regulation is Aurora B, a mitotic kinase that phosphorylates kinetochore substrates to promote
microtubule turnover. The primary Aurora B target for this regulation is the Hec1 subunit of the
NDC80 complex, the primary force-transducing link between kinetochores and microtubules.
Phosphorylation of Hec1 tunes kinetochore-microtubule affinity in cells and allows for precise
graded regulation of attachment stability. While Aurora B is regarded as the “master regulator”
of kinetochore-microtubule attachment stability, whether it works alone or in concert with other
kinases to phosphorylate Hec1 remains unknown. Here we show that Aurora A kinase, which
is implicated primarily in spindle pole function, phosphorylates multiple sites on Hec1 and plays
a key role in regulating kinetochore-microtubule stability. Using phospho-specific antibodies
and small molecule kinase inhibitors, we demonstrate that Aurora A not only contributes to
kinetochore phosphorylation of pole-proximal chromosomes, but surprisingly, that sustained
Aurora A kinase activity is required for regulation of kinetochore-microtubules of aligned,
metaphase chromosomes. Furthermore, we identify serine 69 of Hec1 as the critical Aurora A
target site for regulating kinetochore-microtubule turnover. These findings reveal that both
Aurora B and Aurora A regulate kinetochore-microtubule attachments and importantly, they
uncover an unexpected role for Aurora A kinase in mitosis.
12 11:00-11:20a
Lydia Heasley (CU – Anschutz)
Sporulation is the developmental process by which diploid yeast undergo meiotic divisions and
package each haploid genome into a stress-resistant spore. During the switch from vegetative
(mitotic) growth to sporulation, many macromolecular complexes are remodeled to serve
specialized functions.The complexes formed by the septin proteins are an example of such
remodeling. During mitotic growth, septins localize to the mother-bud neck, where they
facilitate cell division by functioning as protein scaffolds and membrane diffusion barriers
involved in membrane remodeling and directed cell wall synthesis.These roles are carried out
by hetero-octameric septin complexes composed of Cdc3, Cdc10, Cdc11, Cdc12, and Shs1.
However, during sporulation, Cdc12 and Shs1 are excluded by a unknown mechanism and
replaced by the sporulation-specific septins Spr3 and Spr28. These septin complexes form a
series of structures near the growing prospore membrane and are ultimately deposited around
the mature spore membrane. Functional roles for septins during sporulation are poorly defined
in the literature due to discrepancies in reported phenotypes.We have found that the septins
are critical for two key aspects of spore morphogenesis: directed extension, growth, and
curvature of the prospore membrane, and proper deposition of spore wall components. Our
results strongly suggest that the septins are an important component of the spore
morphogenesis pathway.
13 11:20-11:40a
Andy Hoenger (CU – Boulder)
Structure, function and microtubule interaction patterns of hetero-dimeric kinesins
While most kinesins form homodimers or remain monomers, kinesin-2 family members as well
as some kinesin-14 members form heterodimeric structures. In the cases of the yeast
Kar3Vik1 or Kar3Cik1 a motor domain (Kar3) forms a heterodimer with a motor homology
domain (Vik1 or Cik1). We have studied these complexes and their interaction with
microtubules and found them structurally and functionally to be very similar to homodimeric
kinesin-14’s such as ncd. In collaboration with the labs of Susan Gilbert (RPI Troy, NY) and
Ivan Rayment (Univ. of Wisconsin, Madison) we now we are shifting our focus on another
group of heterodimeric kinesins, kinesin-2. Unlike the kinesin-14’s kinesin-2’s are plus-end
directed. For most of the known kinesin-2 members a so-called A-chain can dimerize with a Bchain or a C-chain, depending on location and situation. This creates a set of challenges that is
very different from most other kinesins. We expect to see strong implications in the form of
microtubule interactions as well as their walking dynamics. In particular the mouse kinesin-2C
chain features some new residue sequences that suggest a very different walking mechanism
along microtubules.
14 11:40-12:00p
Dinah Loerke (Univ of Denver)
Biomechanics of cell intercalation
The elongated body axis of most organisms is formed during an early stage of development
through convergent extension, where the tissue narrows in one direction while simultaneously
elongating in the other; one of the primary mechanisms for this process is cell intercalation.
During cell intercalation of the germ band epithelium in the Drosophila embryo, the prevailing
mechanistic model presumes that the driving force for AP junction contraction is unbalanced
increased line tension in AP junctions generated by planar-polarized apical actomyosin
contraction. Using large-scale computational 3D analysis of intercalating cells in the early
embryo, we determined cell dynamics in both the planar and apical-basal axis. Our quantitative
analysis reveal several surprising features about cell intercalation in the Drosophila embryo.
We find that contraction of junctions and T2 formation can initiate from any point along on the
apical-basal axis, including basolateral regions several microns away from the apical cap that
host the major Myosin II populations. We additionally demonstrate an absence of significant
long-range coupling of node movements. These results run counter to expectations based on
the prevailing myosin-centric line tension models, and instead suggest that junction remodeling
occurs through independent ratchet-like sliding step displacements of nodes.
15 4:00-4:20p
Diego Krapf (CSU)
A broad range of membrane proteins display anomalous diffusion on the cell surface. Different
methods provide evidence for obstructed subdiffusion but the structure inducing anomalous
diffusion has never been visualized due to experimental challenges. In particular, membrane
compartments exhibit a dynamic behavior which contributes to the complex diffusion of
membrane proteins, and the resolution needed to observe such structures is beyond the
optical diffraction limit. We image the cortical actin with 40 nm resolution for continuous periods
over more than one minute, while we simultaneously track individual membrane proteins that
interact with the actin cytoskeleton. Our results using dynamic super‐resolution imaging and
single‐particle tracking show that actin introduces barriers leading to compartmentalization of
the plasma membrane and that membrane proteins are transiently confined within actin
domains. Furthermore, our data show that the actin‐induced compartments are scale free and
that the actin cortex itself forms a self similar fractal structure. Thus, compartmentalization
takes place in time scales from milliseconds to several seconds leading to subdiffusion over a
broad timescale, as expected from diffusion in a percolation cluster. These results present a
new nanoscale picture of the plasma membrane and demonstrate scale‐free interactions
between the actin cortex and the cell surface.
16 4:20-4:40p
Meredith Betterton (CU – Boulder)
Kinetochore movements and mitotic spindle dynamics in fission yeast kinesin-8
mutants
Kinesin-8 proteins are plus end-directed motor enzymes that can alter microtubule dynamics
and affect chromosome movements in mitosis, but the mechanisms underlying their mitotic
phenotypes are not yet clear. To better understand the roles of kinesin-8 proteins in mitosis,
we have studied the effects of fission yeast klp5/6 deletions on chromosome movements and
spindle dynamics. We used time-lapse fluorescence microscopy of wild-type and klp5/6
deletion mutant strains and tracked 3D positions of spindle poles bodies and a kinetochore
marker to quantify the dynamics of kinetochore motion and mitotic spindle length. In kinesin-8
deletion mutants we observed new phenotypes in kinetochore-microtubule attachment,
aberrant kinetochore movements away from spindle-pole bodies, delays in chromosome
biorientation, and errors in both kinetochore motion and chromosome segregation. We have
also quantified a striking instability in metaphase spindle length. In contrast to previous studies,
we found significant differences between klp5 and klp6 deletion mutants. We discuss possible
mechanisms driving these cell behaviors, including the idea that kinesin-8 deletion can cause a
weakening or loss of spindle length stabilization.
17 4:40-5:00p
Jim Bamburg (CSU)
A prion-dependent signaling pathway in neuronal oxidative stress and
neurodegenerative disorders.
In a wide variety of stressed cells, actin undergoes dynamic remodeling into rod-shaped cofilinsaturated actin filament bundles (rods). These rods in hippocampal and cortical neurons
occlude neurites, blocking transport, and sequestering cofilin, compromising actin remodeling
associated with synaptic plasticity. Rod formation requires production of reactive oxygen
species (ROS). At least two distinct pathways for ROS production drive rod formation: a
mitochondrial pathway and a cellular prion protein (PrPC)-dependent pathway. Mitochondrial
derived ROS results from neuronal exposure to electron transport inhibitors, hypoxic/ischemic
injury, and excitotoxic glutamate or AMPA. These rods form within 30 min in almost every
neuron and neurite, suggesting rod formation may be associated with stroke. Physiologically
relevant forms and amounts of dementia-inducing β-amyloid peptide (Aβ1-42),
proinflammatory cytokines (e.g. TNFα), and the HIV envelope protein gp120 produce ROS via
the PrPC-dependent pathway requiring NADPH oxidase (NOX). These rods form slowly (half
maximal response in 6 h) and only in about 25% of neurons. In model systems rod formation
leads to synapse dysfunction; thus rods likely have a role in dementia associated with
Alzheimer’s disease, neuroinflammatory syndromes such as traumatic brain injury, and AIDS.
Overexpression of EGFP-PrPC drives rod formation even in the absence of additional factors,
suggesting that PrPC-enriched membrane domains are sites for ROS production. Rods are
found in mouse AD model systems and crossing of these animals with mice in which rod
formation has been inhibited, blocks the learning deficits associated with the AD mice.
18 Poster Session
Location: Gold A120 (Wood Classroom)
**Poster size is limited to 3 x 4 feet (if you can bring thumbtacks it would be appreciated)
POSTER #
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
NAME
Cassi
Estrem
Richard
McIntosh
Jianli
Shi
Philip
Spear
Michele
Jones
Jayne
Aiken
Shannon
Burns
Lynn
Andreas
Andrew
Weems
Carolina
Daez
Kristin
Dahl
Daniel
Sietsema
Lydia
Heasley
Divya
Ganapathi-Sankaran
Kathryn
Wall
Lindsay
Lammers
Ryan
Holly
Cayla
Jewett
Eric
Tauchman
Hui-Shun
Kuan
Bonnie
Bullock
Brian
Bayless
19 23
24
25
26
27
28
29
Alex
Stemm-Wolf
Domenico
Galati
Keith
DeLuca
Westley
Heydeck
Paul
Mooney
Kari
Ecklund
Yi
Xie
20 NEW PRODUCTS FROM CYTOSKELETON
21 22 23