Regular Abstracts - Molecular Biology of the Cell

1490 Pages·2012·9.32 MB·English

SUNDAY-ORAL PRESENTATIONS

ORAL PRESENTATIONS

SUNDAY, DECEMBER 16

Symposium 1: Cell Fate Decisions
1
Lgr5 Stem Cells in self-renewal and cancer.
H. Clevers1; 1Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences & University
Medical Centre Utrecht, Utrecht, Netherlands

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We
originally defined Lgr5 as a Wnt target gene, transcribed in colon cancer cells. Two knock-in
alleles revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. Using
an inducible Cre knock-in allele and the Rosa26-LacZ reporter strain, lineage tracing
experiments were performed in adult mice. The Lgr5+ve crypt base columnar cells (CBC)
generated all epithelial lineages throughout life, implying that it represents the stem cell of the
small intestine and colon. Similar obserations were made in hair follicles and stomach
epithelium.
Single sorted Lgr5+ve stem cells can initiate ever-expanding crypt-villus organoids in 3D culture.
Tracing experiments indicate that the Lgr5+ve stem cell hierarchy is maintained in these
organoids. We conclude that intestinal crypt-villus units are self-organizing structures, which can
be built from a single stem cell in the absence of a non-epithelial cellular niche. The same
technology has now been developed for the Lgr5+ve stomach stem cells.
Intestinal cancer is initiated by Wnt pathway-activating mutations in genes such as APC. As in
most cancers, the cell of origin has remained elusive. Deletion of APC in stem cells, but not in
other crypt cells results in progressively growing neoplasia, identifying the stem cell as the cell-
of-origin of adenomas. Moreover, a stem cell/progenitor cell hierarchy is maintained in early
stem cell-derived adenomas, lending support to the “cancer stem cell”-concept.
Fate mapping of individual crypt stem cells using a multicolor Cre-reporter revealed that, as a
population, Lgr5 stem cells persist life-long, yet crypts drift toward clonality within a period of 1-6
months. Lgr5 cell divisions occur symmetrically. The cellular dynamics are consistent with a
model in which the resident stem cells double their numbers each day and stochastically adopt
stem or TA fates after cell division. Lgr5 stem cells are interspersed between terminally
differentiated Paneth cells that are known to produce bactericidal products. We find that Paneth
cells are CD24+ and express EGF, TGF-a, Wnt3 and the Notch ligand Dll4, all essential signals
for stem-cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells
dramatically improves organoid formation. This Paneth cell requirement can be substituted by a
pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitantloss
of Lgr5 stem cells. In colon crypts, CD24+ cells residing between Lgr5 stem cells may represent
the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche
signals provided by a specialized daughter cell, the Paneth cell.

2
Gene Regulatory Networks Governing Hematopoietic Stem Cell Development and
Identity.
T. Enver1; 1UCL Cancer Institute, University College London, London, United Kingdom

Several studies have addressed questions about transcriptional regulation within particular
hematopoietic cell compartments. Few, however, have attempted to capture the transcriptional
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changes that occur during the dynamic transition from one compartment to another. We have
profiled gene expression as multipotential progenitors underwent commitment and
differentiation to two alternative lineages, focusing on the first 3 days of differentiation when the
majority of decisions about cell fate are made. We have combined this with genome-wide
identification of the targets of three key transcription factors before and after differentiation;
GATA-2, usually associated with the stem/progenitor compartment; GATA-1 (erythroid); and
PU.1 (myeloid). We used correlation analyses to associate transcription factor binding with
particular modules of co-expressed genes, alongside detailed sequence analysis of bound
regions. Dynamic modelling of TF relationships has predicted novel interactions that have been
validated experimentally. These approaches have highlighted novel regulators of stem cell fate
decisions and - informed our understanding of GATA factor switching. Overall, the data reveal
greater degree of complexity in the interplay between GATA-1, 2 and PU.1 - in regulating
hematopoiesis than has hitherto been described, and highlights the importance of a genome-
wide approach to understanding complex regulatory systems. A significant challenge in the field
is how to relate these types of population-based data to the action of transcriptional regulators
within single cells where cell fate decisions ultimately are effected. As a step toward this, we
have generated single cell profiles of gene expression for a limited set of transcriptional
regulators in self-renewing and committed blood cells and used these data to build a stochastic
computational model, which affords exploration of commitment scenarios in silico. The data
highlight the ‘noisiness’ of transcription in multipotential cells, and we have computationally
captured modes of regulation that may contribute to heterogeneous gene expression. We
suggest that individual cells may enter lineage commitment through different routes; data on
instruction of commitment through perturbation of individual regulators is in support of this view.

3
Asymmetric cell division and spindle orientation in neural stem cells - from Drosophila to
humans.
Juergen A. Knoblich1; 1Institute of Molecular Biotechnology, Austrian Academy of Sciences,
Vienna, Austria
When we think of mitosis, we commonly have a process in mind where a cell gives rise to two
identical daughter cells. In whole organisms, however, many cell divisions are actually
asymmetric and give rise to two daughter cells of different size, shape or developmental fate.
Asymmetric cell divisions are particularly important in stem cells, as they allow those cells to
generate both self-renewing and differentiating daughter cells, an ability that is common to all
stem cells. We therefore use stem cells in the developing brain of both fruitflies and mice as a
model to understand the principle mechanisms that regulate and orient asymmetric cell
divisions. More recently, we have extended our efforts to mammalian model systems, where
mutations in regulators of basic cell biological processes like the orientation of the mitotic
spindle are known to cause strong brain malformations resulting in severe mental retardation.
As recent experiments have shown striking differences between human and mouse brain
development, we have made an effort to establish experimental strategies where those
regulators and their effects on brain development can be studied in a human setting.

Bruce Alberts Award for Excellence in Science Education

4
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International Institute for Collaborative Cell Biology and Biochemistry (IICCBB). Building
a network to share and inspire.
L. C. Cameron1; 1Laboratorio de Bioquimica de Proteinas, Universidade Federal do Estado do
Rio de Janeiro, Rio de Janeiro, Brazil

In 2000 we organized the first international symposium on myosin V in my home city of Rio de
Janeiro. That meeting became part of the foundation for a series of courses focused on hands-
on research training and education workshops to train the next generation of international
scientists, with particular emphasis on Central and South American students who have limited
opportunities to interact directly with international scientists. My colleagues and I founded The
International Institute for Collaborative Cell Biology and Biochemistry (IICCBB). The IICCBB is a
network of world experts who want to share their expertise and knowledge to inspire students,
who in future will be the scientific leaders in the respective fields of biochemistry, cell biology
and biotechnology. Our vision is to invigorate a new generation of international scientific
cooperation by exposing young scientists to diverse, multidisciplinary learning experiences via
organizing various workshops, conferences and symposia. At these events, leading established
scientists connect with each other and share their wealth of experience with the next generation
of scientists who, in turn, act as ambassadors to their colleagues. We organized more than 25
of these workshops, international conferences and courses in Brazil, Uruguay, Mexico, and the
United States (we had more than 1600 attendees). Subject areas include topics in cell biology
(calcium signaling, intracellular transport, and other aspects of the cytoskeleton), biochemistry,
biophysics, and systems biology. These training courses have had a great impact on the
students, exposing them to North American and European science. Many have gone on to work
in the laboratories of the U.S. and European faculty who have participated, and the courses
have sparked multiple intercontinental collaborations. We are now planning to expand to other
regions of Brazil and Americas and to motivate colleagues to promote courses in other Latin
America countries, Africa and Asia. In addition, we are working to consolidate the IICCBB with
more dependable funding from national and international agencies. We hope that we can
continuous grow a friendly network of science all over the world.

E.E. Just Lecture

5
Decoding the biology of human genome polymorphisms in African Americans.
G. M. Dunston1,2, T. Mason2, J. Lindesay3; 1Microbiology, Howard University, Washington, DC,
2National Human Genome Center, Howard University, Washington, DC, 3Computational Physics
Laboratory, Howard University, Washington, DC

The completion of the Human Genome Project introduced a new knowledge system for
decoding biology based on the science of information structured in DNA sequence variation.
Single nucleotide polymorphisms (SNPs) are a system of common variation widely distributed
across the genome, where two or more forms of the DNA sequence are found at a given site,
with the rarest form occurring at a frequency of one percent or greater in the population.
Recently, our Biophysics Research and Development Group has applied first principles of
thermodynamics and statistical physics in studying the informatics of SNPs, as dynamic sites in
the genome. From our perspective of SNPs as complex dynamical systems, we derived a new
biophysical metric for interrogating the information content (IC) present in SNP haploblocks.
This metric facilitates translation of biochemical sequence variation into a biophysical metric
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derived from Boltzmann’s canonical ensemble used in information theory. Our normalization of
this information metric (NIC) allows for comparison of SNP haploblocks across different sites in
the genome. We found that low NIC scores in the human leukocyte antigen-disease related
(HLA-DR) region, illumined molecular pathways of innate immune mechanisms functional in
host adaptation to environmental stressors, such as pathogens. In our approach, we were able
to relate NIC to biologically relevant functional knowledge embedded in the structure of common
sequence variation. We are exploring new perspectives of human population biology interfacing
genomics and theoretical physics at the frontier of life sciences.

Minisymposium 1: Cancer Cell Biology

6
In vivo imaging of dynamic interactions within the haematopoietic stem cell niche.
C. Lo Celso1; 1Imperial College London, London, United Kingdom

Haematopoietic stem cells (HSC) reside within the bone marrow, therefore their
microenvironment (or niche) has been traditionally inaccessible to direct observation. Despite
numerous functional studies demonstrating that several bone marrow stroma cells regulate HSC
function and number, still very little is known about the anatomical location of HSC during
steady state and in response to several types of stimuli, including leukaemia development. It is
an open question whether HSC firmly localise within a specific niche or whether they migrate
between different niches, defined by distinct bone marrow microenvironments, where they
receive different stimuli. Other open questions are whether leukaemia development affects
HSC-niche interactions and whether leukaemia stem cells (LSC) and HSC compete for niche
space.

Confocal/two-photon hybrid microscopy allows visualization at single cell resolution of
transplanted haematopoietic stem and progenitor cells (HSPC) in the calvarium bone marrow of
live mice. FACS-purified, ex-vivo labelled HSPC populations can be visualised upon their initial
arrival in the niche (homing) and following the first divisions (early engraftment). We showed
that long-term repopulating (LT) HSPC selectively localize proximal to osteoblasts within a few
hours from transplantation, whereas their progeny are more distal and we collected further
evidence that LT-HSPC and their progeny localization near osteoblasts correlates with positive
transplantation outcome. Finally, we used a mouse model of acute myeloid leukaemia based on
retroviral transduction of the MLL-AF9 oncogene and observed that leukaemic cells
corresponding to different stages of disease progression follow distinct homing patterns. In
particular, granulocyte-monocyte precursors (L-GMP), an established LSC population, share the
same homing pattern of normal GMP rather than that of LT-HSPC. This indicated why MLL-AF9
LSC are insensitive to niche-derived signals that otherwise affect normal HSC function.

7
Autophagy dependent secretion of interleukin-6 facilitates cancer cell invasion.
J. Debnath1, R. Lock1, C. Kenific1, E. Salas1; 1Pathology, University of California, San Francisco,
San Francisco, CA

To date, the pro-tumor functions of autophagy have been largely attributed to its ability to
promote tumor cell fitness and survival. We now demonstrate a new role for autophagy
regulators (ATGs) in facilitating tumor cell motility, extracellular matrix invasion, and in vivo
metastatic capacity. In Ras-transformed epithelial cells, RNAi-mediated depletion of ATGs
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profoundly inhibits invasive behavior in three-dimensional (3D) culture and restores multiple
aspects of normal epithelial architecture, including polarized deposition of basement membrane
and cell-cell junctional integrity. Furthermore, lung metastases in vivo are attenuated upon
autophagy inhibition in Ras-transformed epithelial cells and in Polyoma Middle T (PyMT) breast
tumor cells.

Importantly, the invasion defect in autophagy-deficient cells is completely rescued upon
treatment with conditioned media from autophagy-competent counterparts, indicating that intact
autophagy is required for the elaboration of secreted pro-invasive factors. In support, we identify
the pro-invasive cytokine interleukin-6 (IL6) as one of the critical factors whose secretion is ATG
dependent. Upon ATG knockdown, Ras-transformed cells fail to secrete IL6 into the conditioned
media, although both IL6 transcription and translation remain intact. Moreover, function-blocking
studies and rescue experiments using recombinant IL6 substantiate that this cytokine is both
necessary and sufficient to restore invasion in autophagy-deficient cells. In addition to IL6,
autophagy-defective cells exhibit reduced levels of other pro-invasive molecules, including
Wnt5a and matrix metalloproteinase 2 (MMP2). Addition of Wnt5a partially rescues the invasion
defect in autophagy depleted H-RasV12 cells, while pharmacological MMP-2 inhibition potently
suppresses invasion. Overall, these results support that autophagy mediates the coordinate
production of multiple secreted factors that favor invasion in oncogenic epithelial cells. They
also point to a broader role for autophagy in carcinoma progression, namely facilitating invasion
during dissemination and metastasis.

8
GATA3 suppresses metastasis, promotes differentiation and modulates the tumor
microenvironment by regulating microRNA-29b expression.
J. Chou1, J. Lin1, A. Brenot1, J-W. Kim1, S. Provot1, Z. Werb1; 1Anatomy, Univ. of California, San
Francisco, San Francisco, CA

Despite advances in our understanding of breast cancer, patients with metastatic disease have
poor prognoses. GATA3 is a transcription factor that specifies and maintains mammary luminal
epithelial cell fate, and its expression is lost during breast cancer progression. Indeed, low
GATA3 expression correlates with poor prognosis in human patients. Here, using human and
mouse breast cancer cells, we show that GATA3 promotes differentiation, suppresses
metastasis and alters the tumor microenvironment by inducing miR-29b expression. Consistent
with miR-29b being downstream of GATA3, miR-29b is enriched in better prognostic, luminal
breast cancers and increases luminal gene expression. Mechanistically, miR-29b inhibits
metastasis by directly targeting a network of pro-metastatic regulators involved in angiogenesis,
collagen remodeling and extracellular matrix proteolysis, including VEGF, ANGPTL4, PDGF,
LOX and MMP9. In addition, miR-29b targets TGFB, thereby indirectly regulating epithelial
plasticity. Loss of miR-29b, even in GATA3-expressing cells, increases metastasis and
promotes a mesenchymal phenotype. This discovery that regulating tumor microenvironmental
genes and differentiation through a GATA3-miR-29b axis inhibits cancer metastasis opens up
new possibilities for therapeutic intervention in breast cancer.

9
aPKC iota/lambda regulates Hh signaling during basal cell carcinoma growth.
S. X. Atwood1, M. Li1, J. Y. Tang1, A. E. Oro1; 1Dermatology, Stanford University School of
Medicine, Stanford, CA

Basal cell carcinoma (BCC) initiation and expansion requires high levels of Hedgehog (Hh)
signaling. While Smoothened (Smo) inhibitors are effective, early tumor resistance illustrates the
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need for additional downstream targets for therapy. Here we identify atypical Protein Kinase C
iota/lambda (aPKC) as a novel BCC oncogene essential for Hh signaling. Genetic knockdown
using shRNA against aPKC, or pharmacological inhibition of aPKC, inhibits proliferation, Hh
signaling, and ciliogenesis of the murine BCC cell line ASZ001. Genome-wide transcriptome
analysis of BCC cells reveals pharmacological inhibition of aPKC or Smo target similar
pathways. aPKC is a Hh target gene that is overexpressed in mouse and human BCCs and
functions downstream of Smo to bind and phosphorylate Gli1, resulting in maximal DNA binding
and Hh activation. Consistent with its role in regulating Hh signaling, application of a topical
aPKC inhibitor suppresses Hh signaling and tumor growth in primary murine BCC tumors. As
acquired drug resistance is a growing problem, we also demonstrate Smo antagonist-resistant
BCC cells and human tumors overexpress active aPKC and pharmacological inhibition of aPKC
suppresses proliferation. These results demonstrate polarity signaling is critical for Hh-
dependent processes and suggest aPKC may be a new therapeutic target for the treatment of
naïve and resistant BCCs.

10
BRAFV600Eand PI3’-kinase signaling pathways cooperate to regulate protein translation in
human melanoma cells.
J. M. Silva1, C. Bulman1, M. McMahon1; 1Helen Diller Family Comprehensive Cancer Center and
Department of Cellular and Molecular Pharmacology, University of California, San Francisco,
San Francisco, CA

The most common genetic alteration in metastatic melanoma is a T1799A transversion that
encodes BRAFV600E resulting in the constitutive activation of the BRAF→MEK→ERK MAP
kinase pathway. In many cases, the conversion of BRAFV600E expressing melanocytes to
melanoma cells also requires activation of the PI3’-kinase signaling pathway, which can occur
through the silencing of the tumor suppressor PTEN, a PI3’-lipid phosphatase. To explore the
mechanisms of cooperation between the BRAFV600E and PI3’-kinase signaling pathways, we
applied pharmacological inhibitors of each pathway to BRAFV600E expressing human melanoma-
derived cell lines. Our data demonstrated that blockade of BRAFV600E→MEK→ERK signaling
inhibited the phosphorylation of the cap-dependent translational regulators, p70S6K, ribosomal
protein S6 (rpS6), and 4EBP1, whereas inhibition of PI3’-kinase only suppressed rpS6 and
4EBP1 phosphorylation. In addition, blockade of BRAFV600E→MEK→ERK or PI3’-kinase also
led to the inhibition of melanoma cell proliferation. Moreover, simultaneous inhibition of both
BRAFV600E and PI3’-kinase signaling showed that these two pathways cooperate to regulate
protein synthesis through mTORC1-dependent effects on rpS6 and 4EBP1. However,
pharmacological inhibition of the downstream PI3’-kinase effector, AKT, only had modest effects
on cell growth and p70S6K, rpS6, or 4EBP1 phosphorylation, even in melanoma cells that
contain mutated or amplified AKT. Taken together, these data suggest that BRAFV600E and PI3’-
kinase cooperate to regulate melanoma proliferation and protein translation independent of AKT
activation and combined targeting of BRAFV600E and PI3’-kinase signaling may help to enhance
the therapeutic efficacy in patients with mutations that activate both of these pathways.

11
Widespread potential for growth factor-driven resistance to anti-cancer kinase inhibitors.
J. Settleman1, T. Wilson1; 1Genentech, South San Francisco, CA

Mutationally-activated kinases, including EGFR, HER2, ALK, ABL, PDGFR, c-KIT, and BRAF,
define a clinically validated class of targets for cancer drug therapy. However, the efficacy of
kinase inhibitors in patients whose tumors harbor such alleles is invariably limited by innate or
acquired drug resistance. As molecular mechanisms of resistance have begun to be elucidated,
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a recurrent theme is the engagement of survival signals redundant to those transduced by the
targeted oncogenic kinase. Cancer cells typically express multiple receptor tyrosine kinases
(RTKs), and many of these can potentially mediate signals that converge on common and
critical downstream cell survival effectors - most notably, PI-3 kinase (PI3K) and MAP kinase
(MAPK). Consequently, a change in the production or availability of ligands for such RTKs,
either through autocrine tumor cell production or paracrine contribution by tumor stroma, could
confer resistance to inhibitors of an oncogenic kinase with a similar downstream signaling
output. Indeed, using a large panel of kinase-“addicted” cancer cell lines, we found that the vast
majority of such cells can be “rescued” from drug sensitivity by simply exposing them to one or
more RTK ligands that engage redundant survival effectors. Among the findings with immediate
clinical implications was the observation that hepatocyte growth factor (HGF), which is widely
expressed in tumour stroma, confers resistance to the BRAF inhibitor PLX4032 in BRAF mutant
melanoma cells, and to the HER2 kinase inhibitor lapatinib in HER2 amplified breast cancer
cells. These observations highlight the extensive redundancy of RTK-transduced signalling in
cancer cells and the potentially broad role of widely expressed RTK ligands in both innate and
acquired resistance to drugs targeting oncogenic kinases.

Minisymposium 2: Cell Mechanics and Intermediate Filaments

12
From isolated filaments to polymer-bundles in cells.
S. Koester1; 1Institute for X-Ray Physics, Georg-August-University Goettingen, Goettingen,
Germany

The polymeric nature of intermediate filaments (IF) leads to a great variety of intracellular
structures which determine mechanical properties and thereby the diverse cytoskeletal
functions. In order to understand the physical basis of the phenomena that are observed in
cells, we combine in vitro experiments involving purified protein with cell studies.
In a bottom-up approach, the mechanical rigidity of individual IFs, characterized by their
persistence length, is measured while taking into account confinement effects which cellular
components encounter in vivo. In a cell, however, the situation is more complex since the
individual IFs form networks of bundles and thereby interactions within and between such
supramolecular assemblies have to be taken into account. The structure of the bundles is
assessed by novel X-ray nanodiffraction methods while the dynamics are captured by
fluorescence life-cell imaging. All the above mentioned experiments are performed in
specifically tailored microfluidic sample environments. By this approach we are able to mimic
and precisely control close-to-physiological conditions

13
The effect of small heat shock proteins on intermediate filament networks.
J. Kayser1, M. Haslbeck2, H. Grabmayr1, J. Buchner2, H. Herrmann3, A. R. Bausch1; 1Physics
Departement, Technische Universität München, Garching, Germany, 2Department of Chemistry,
Technische Universität München, Garching, Germany, 3Deutsches Krebsforschungszentrum,
Heidelberg, Germany

Despite their importance and abundance, very little is known about the physics of intermediate
filaments. The largest subgroup of the intermediate filament protein family comprises keratins,
with 28 type I (acidic) and 26 type II (basic) keratins. Their principal assembly subunit is a
heterodimeric coiled-coil complex of one acidic and one basic keratin. Due to the antiparallel
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fashion in which these dimers connect to tetrameric complexes, the basic module for filament
assembly, the resulting filaments are apolar and exhibit a surprising degree of dynamics and
restructuring within cells. While the assembly kinetics in the early stages of filament assembly
has been addressed, their growth and association into filamentous networks is still an enigmatic
process. By in vitro studies with reconstituted 8/18 keratins, we demonstrate an inherent
tendency to interact with each other under physiological salt conditions. The structure of the
resulting network is determined by the competition of filament elongation and lateral association
to bundles. Small heat shock proteins modulate the association kinetics and cause a drastic
alteration of network structure and morphology. This seems to be an essential tool for cells to
regulate the organization of their intermediate filament cytoskeleton.

14
Vimentin dynamics and microtubule crosstalk during fibroblast migration.
J. D. Tytell1, L. Ding1, N. Costigliola1, J-H. Su1, G. Danuser1; 1Cell Biology, Harvard Medical
School, Boston, MA

Overexpression of the intermediate filament protein vimentin is a key biomarker for metastasis.
Conversely, fibroblasts lacking vimentin expression are defective in wound healing and in
maintaining persistent, directional migration. These data suggest that vimentin might play an
important role in regulating cell migration. However, very few studies have addressed the
dynamics and function of vimentin polymers in migrating cells. One clue to vimentin’s role in
migration comes from its interactions with microtubules, another cytoskeletal polymer that
establishes and maintains cell polarity. It has been documented that vimentin assembly
depends on microtubules and microtubule dependent motor proteins. However, whether
vimentin affects microtubule organization and dynamics in migrating cells is unknown. To
address these questions, we investigated vimentin dynamics in randomly migrating fibroblasts
expressing mEmerald-Vimentin. We found that vimentin is a highly dynamic filament on a
slightly longer time scale than microtubules. Vimentin filaments are oriented parallel to the
direction of migration, are recruited quickly into the leading edges of migrating cells and
dynamically cycle from the exterior to the interior of the cells even in stationary cells. Therefore,
contrary to previous belief, vimentin is a highly dynamic polymer that changes structure
concurrent with cell movement. The fact that vimentin localizes dynamically to the same places
as microtubules during migration but on a slightly longer timescale suggested that vimentin
might help stabilize the direction of movement perhaps by feeding back to microtubules. In order
to analyze the dynamics of vimentin and microtubules simultaneously, we tracked microtubule
growth by following microtubule plus ends over time. We find that a subset of dynamic
microtubules follow along vimentin tracks and another subset follow vimentin towards the
leading edge and then continue past the vimentin track into the leading edge of cells. From
these data, we hypothesize a template model whereby microtubules enter the leading edge first
and recruit vimentin fibers, which in turn serve as a support for the more dynamic microtubules
to continue growth in that direction, thus establishing a positive feedback loop leading to
increased memory of cell polarity. Without vimentin, microtubules would still establish polarity
but lose polarity more rapidly leading to decrease in migration in a given direction.

15
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Cell stiffness correlates with cell volume.
M. Guo1, E. Zhou2, F. Mackintosh3, J. Fredberg2, J. Lippincott-Schwartz4, D. Weitz1; 1SEAS,
Harvard University, Cambridge, MA, 2Medical School, Harvard University, Cambridge, MA,
3Physics, Vrije Universiteit, Amsterdam, Netherlands, 4Cell Biology and Metabolism, National
Institutes of Health, Bethesda, MD

Cells usually have a certain size, and their size has been shown to be correlated to gene
expression across species. There are careful studies about standard cell sizes of different
species, as well as cell size changes during stages of the cell cycle. Furthermore, people have
shown that cells change their spreading area, stiffness, gene expression and even stem cell fate
when grown on substrates of varying stiffness. However, whether cell size changes for the
same cell type under varying micromechanical environments, and whether this is related to the
observed phenomenological changes is not known. Here we show that height and volume of
single adherent cells decrease when grown on a stiffer 2D substrate, while spreading area
increases. When cell spreading area is confined by micropatterning on stiff substrate, we find
cell volume is dependent inversely on their spreading area. We further measure cell stiffness
with optical magnetic twisting cytometry. By controlling their volume in three different ways –
varying substrate stiffness, cell spreading areas, and osmotic pressure in the medium – we find
that cell stiffness correlates with cell volume but not substrate stiffness. Cells can be soft on a
stiff substrate by changing only the spreading area; they can be stiff on a soft substrate by
increasing osmotic pressure. Futhermore, we show that vimentin may be involved in cellular
volume regulation.

16
Lamin mutations that cause muscle defects disturb nuclear mechanics and nucleo-
cytoskeletal coupling
M. Zwerger1, D. E. Jaalouk2, M. Lombardi3, M. Mauermann4, H. Herrmann4, L. L. Wallrath5, J.
Lammerding6; 1Department of Biochemistry, University of Zurich, Zurich, Switzerland, 2American
University of Beirut, Beirut, Lebanon, 3Brigham and Women's Hospital, Boston, MA, 4German
Cancer Research Institute, Heidelberg, Germany, 5University of Iowa, Iowa City, IA, 6Biomedical
Engineering/Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY

Objective: Mutations in the LMNA gene that encodes the nuclear envelope proteins lamin A
and C cause a plethora of human diseases (laminopathies), including muscular dystrophies,
cardiomyopathies, and familial partial lipodystrophy. It remains unclear how mutations in a
single gene that is ubiquitously expressed result in such often tissue-specific diseases. Since
lamins A and C are the main contributors to nuclear stiffness, we hypothesized that lamin
mutations associated with muscular phenotypes could impair the structural properties of the
nuclear lamin network, weakening the nuclear integrity and resulting in cells more susceptible
to mechanical stress.
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Methods: We measured nuclear stiffness in fibroblasts derived from patients with diverse
laminopathies and in lamin A/C–deficient mouse embryonic fibroblasts (MEFs) engineered to
stably express physiological levels of specific lamin A mutants. In a subset of cells, we also
probed nucleo-cytoskeletal coupling with a custom-developed microneedle assay. To address
the effect of diverse lamin mutations on the ultrastructural level, we observed the in vitro
assembly of purified mutant and wild-type lamins. To assess the in vivo effect of lamin
mutations in muscle, we measured nuclear stiffness in body wall muscle of Drosophila
melanogaster models of muscle laminopathies.

Results: Patient fibroblasts carrying LMNA mutations associated with muscular dystrophies
had ‘softer’ nuclei than cells from healthy controls. In contrast, fibroblasts from lipodystrophy
patients had normal nuclear mechanics. Extending our studies to MEFs expressing a panel of
lamin A mutations, we found that four of the 15 lamin A mutations tested caused decreased
nuclear stiffness. Importantly, all four mutations were associated with laminopathies affecting
muscle tissue, whereas mutations linked to lipodystrophy had no effect on the structural function
of lamin A. Of note, most mutations linked to muscular disease also showed disturbed nucleo-
cytoskeletal coupling. Extending these studies to muscle tissue, we found that mutant lamins
alter the stiffness of nuclei in isolated Drosophila body wall muscle, where nucleo-cytoskeletal
coupling is also perturbed.

Conclusions: LMNA mutations associated with muscular laminopathies can cause impaired
nuclear mechanics, which is consistent with increased cellular sensitivity to mechanical stress
that could contribute to the muscle-specific phenotypes associated with the laminopathies. In
conclusion, our results demonstrate the importance of lamins A and C on nuclear mechanics in
laminopathies, but also indicate that additional factors such as altered nucleo-cytoskeletal
coupling influence the disease outcome.

17
The nuclear mechanostat that scales with tissue stiffness and amplifies lineage: lamin-
A,C.
J. Swift1, I. Ivanovska1, T. Harada1, J. Pinter2, A. Buxboim1, J-W. Shin1, M. Tewari2, D. W.
Speicher3, D. E. Discher1; 1University of Pennsylvania, Philadelphia, PA, 2Bioengineering,
University of Pennsylvania, Philadelphia, PA, 3Wistar Institute, Philadelphia, PA

Tissue can be soft like brain, stiff like muscle, or rigid like bone. Proteomic profiling of human
and mouse tissues and cells reveals that the nucleoskeletal protein lamin-A,C scales with
various collagens and with tissue microelasticity, E. Among the many cell structure and nuclear
components quantified here, lamin-A,C acts most clearly as a “mechanostat” in increasing as ~
E^0.7, whereas B-type lamins are nearly constant. Lamin-A,C dominates in stiff tissues and has
been implicated in aging and diseases that impact muscle, bone, and fat but rarely brain or
marrow, and nuclei in stiff tissue cells also prove much stiffer than nuclei from softer tissues.
Mesenchymal stem cell differentiation in vitro further shows that lamin-A,C amplifies lineage
signals from matrix, with low lamin-A,C favoring a soft tissue fate and high levels favoring stiff
tissue. Regulation of lamin-A,C occurs at multiple levels, with conformational changes in
isolated nuclei revealing its direct response to stress. Systematic relations thus exist between
tissue stress and stiffness and the nucleus.

Minisymposium 3: Cell Migration and Motility