Projects

Here you will find brief descriptions of specific research projects pursued in the lab. Some have already been completed and published while others are ongoing efforts. There’s still so much more to discover. What would you like to figure out about cytoskeleton dynamics in vivo?

Actomyosin structures and their function in the C. elegans germline syncytium

Syncytial architecture is an evolutionary-conserved feature of the germline across species, from insects to humans, and plays a crucial role in organism fertility. During gametogenesis, germ cells undergo incomplete cytokinesis giving rise to multiple germ cell nuclei connected to each other through stable intercellular bridges sharing a common cytoplasm. Interestingly, these intercellular bridges are enriched in actomyosin regulators. Recent studies suggested actomyosin regulators are important in the stabilization of germ cell intercellular bridges, however the role of contractility in the maintenance of germline structure remains poorly understood.

We are using the Caenorhabditis elegans gonad as a model to study mechanobiological properties of the germline syncytium and the role played by various actomyosin machinery structural components and regulators.

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Role of non-junctional cadherins in regulating the cortex

Several years ago we made the surprising discovery that E-cadherin formed clusters all over the cell membrane, independently of cell-cell junctions. We went on to show that these clusters, which are delimited by cortical F-actin, can become adhesive clusters when they come in contact with another cell and form cell-cell junctions when they congregate at high density. This work was published in Dev Cell 32(2):139-54. However, we were left with an open question: do non-adhesive cadherins have a role outside of cell-cell junctions, aside from being building material for junctions?

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An answer to that question came from experiments we performed in C. elegans. We discovered the nematode ortholog of E-cadherin, HMR-1, also forms non-junctional clusters in the early embryo and they affect cortical dynamics.

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Non-junctional cadherin clusters were found to inhibit Rho-1 and the level of non-muscle myosin II at the cortex. Moreover, non-junctional cadherin increased the friction between cortical actomyosin and the membrane and slowed cortical flows. This resulted in C. elegans cadherin slowing down cytokinesis as can be seen in the following figure:

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Perhaps the most remarkable phenotype we observed with the depletion of HMR-1/E-cadherin in the C. elegans zygotes occured in about 10% of the zygotes: a complete detachment of the actomyosin cortex from the plasma membrane:

This phenotype raises many questions about the regulation of cortical dynamics in the absence of membrane. We are now optimising conditions in which this phenotype will be highly penetrant and robust so we can address those questions.

In parallel, we are also currently investigating the role of mammalian cadherins in regulating cortical dynamics outside of cell-cell junctions. Preliminary results in single mammary epithelial cells indicate that such a role is conserved.

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Regulation of actomyosin contractility in the C. elegans zygote

Cortical contractility, driven by an actomyosin network underlying the plasma membrane, is responsible for the control of cell shape and the generation of contractile forces that drive polarization and cell division. Contractility depends on myosin activity as well as the organization of filamentous actin, which is determined by the concerted action of a multitude of actin binding proteins.

NMY-2 LifeAct Cytokinesis

We have elucidated the role of plastin, an evolutionary-conserved actin-bundling protein (a.k.a fimbrin), in providing the cortex with the required amount of connectivity to generate long-range contractions and coordinated movement (cortical flows). Such connectivity is essential for cell-scale processes, such as polarisation and cytokinesis. Plastin loss of function mutant (tm4255) shows defects in many contractility-dependent processes, including polarisation and cytokinesis.

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Our work on plastin was published in the Journal of Cell Biology in May 2017. We are currently studying the function of other actin binding proteins in regulating cortical contractility in the early embryo and the role of plastin later in development.

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Self organization of the actomyosin cytoskeleton in fibroblasts

We collaborated with Sasha Bershadsky on a project utilising structured illumination microscopy to follow the dynamics of non-muscle myosin IIA in rat embryonic fibroblasts (REF52) cells. We first characterised the structure of stress fibers, resolving the repetitive sarcomere-like unit (see panel G in figure below). Next, we followed the co-assembly of myosin filaments with actin filaments into stress fibers and their further alignment into “myosin stacks”, which are supracellular structures that run perpendicular to the direction of the stress fibers. Various chemical inhibitors and siRNA knockdown were used to demonstrate that the assembly of myosin stacks depends on actin assembly/disassembly dynamics and myosin motor activity. This work was published in Nature Cell Biology 19(2):133-141 doi: 10.1038/ncb3466.

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We are now further studying the proteins and molecular mechanisms involved in myosin IIA stack formation. An siRNA screen of candidate Contractome proteins has led to the discovery of two opposite phenotypes, as shown below: 1. collapse of stress fibers into large bundles that completely loose the order in myosin organization and 2. dramatic enhancement of myosin stack formation, causing the fibroblast to look like muscle cells. We are currently studying the genes that give rise to these strikingly opposing phenotypes.

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Regulation of actomyosin contractility in the C. elegans spermatheca

The C. elegans spermatheca is an accordion-like epithelial tube made of 24 cells, in which fertilization takes place. An oocyte comes in from the gonad side, gets fertilized by sperm that is stored in the spermatheca (hence its name!), and then exists into the uterus as a one cell embryo.You can watch the process of ovulation in this movie:

The spematheca can be found in one of two conformation: collapsed, when it is empty, or stretched, when an oocyte is inside. The relaxed spermatheca expands due to the oocyte being pushed inside and it constricts due to actomyosin contractility.

(images by Limor Broday and Irina Kolotuev, taken from WormAtlas)

The spermatheca cells are known to have actin bundles organized in a circumferential orientation, but how actomyosin contraction is regulated in a cyclical manner is unknown and is the subject of research in our lab.

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We have identified SPV-1, an F-BAR and RhoGAP protein as a novel regulator of spermatheca contractility. Interestingly, its localization to the plasma membrane appears to be curvature dependent and we propose that is acts as a mechanosensor, transducing the mechanical signal of membrane stretching into a biochemical signal, i.e. RhoA activity. Such feedback between membrane curvature and regulation of actomyosin contractility could potentially be at play in other cellular processes, such as cell migration, so there is much more to discover.

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We are currently working on the Rho GEFs that activate Rho-1 in the spermatheca and understanding how they are affected by stretching of the spermatheca. We are also collaborating with Erin Cram‘s lab on the interplay between calcium signalling and Rho-1 signalling in the spermatheca.

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The Contractome

We are very interested in the regulation of actomyosin contractility in non-muscle cells. Actomyosin contractility plays an important role in many cellular processes, from cell division, spreading and migration, through adhesion and modulation of the extracellular matrix, to intracellular transport and nuclear structure and function. In collaboration with Chen Luxenburg from Tel Aviv University (http://www.luxenburglab.com/) we assembled a comprehensive inventory of cellular functions of actomyosin contractility and a parts list of proteins involved in either the structure or regulation of contractility in non-muscle cells. We then used bioinformatics and literature mining to obtain all the known interactions between the different proteins. We nicknamed the resulting network of interacting proteins the Contractome.

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We trust this resource will be useful for teaching and for researchers in the community. It is already proving to be a useful guide in our research into the regulation of contractility in mammalian cells and in C. elegans.

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Formin-mediated Actin Polymerization and Cadherin-mediated cell-cell adhesion [Completed Project]

E-Cadherin-mediated Adherens Junctions (AJs) in epithelial cells are anchoring junctions that help connect cells in a tissue by bridging the cytoskeleton of neighboring cells. Binding to the cytoskeleton allows these junctions to function as force-sensors and participate in morphogenesis, migration, tissue repair and regeneration. The formin family of actin nucleators (that is, proteins that build the actin cytoskeleton) is implicated in generating several cellular actin structures; however, their role at AJs remains poorly characterized. Using mammalian epithelial cells in culture, we elucidated the role of actin polymerization mediated by formins in regulating E-Cadherin clustering and dynamics at AJs. We identifed mDia1 and Fmnl3 as major factors enhancing actin polymerization and stabilizing E-cadherin at epithelial junctions. Fmnl3 localizes to adherens junctions downstream of Src and Cdc42 and its depletion leads to a reduction in F-actin and E-cadherin at junctions and a weakening of cell–cell adhesion. Of importance, Fmnl3 expression is up-regulated and junctional localization increases during collective cell migration. Depletion of Fmnl3 or mDia1 in migrating monolayers results in dissociation of leader cells and impaired wound repair. These results were published in Molecular Biology of the Cell 27(18):2844-56. doi: 10.1091/mbc.E16-06-0429.

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Evolution of the cadherin adhesome [completed project]

When confronted with the immense complexity of the cadherin adhesome it is only natural to wonder how did this complexity arise? Furthermore, if one assumes that such a large network of proteins is essential for the function of cell-cell adhesion and that cell-cell adhesion was essential for the emergence of multicellularity but not needed before hand, then how did this major step in evolution occur?

Remarkably, thanks to the whole genome sequencing of many organisms we now know that the step from unicellular to multicellular actually occurred separately multiple times throughout evolution! (As reviewed in Abedin and King, Trends in Cell Biology Volume 20, Issue 12, 2010, Pages 734–742)

Taking advantage of the abundance of sequenced genomes, in a close collaboration with Paul Murray and Barry Honig (Columbia U. NY, NY), we are exploring the evolution of the cadherin adhesome, from our unicellular origins. It is a fascinating story because you can find a unicellular choanoflagellate that has cadherin but no catenins and an even more ancient slime mold that has catenins but no cadherin. How did they all come together in sea sponges is something we are now figuring out.

UPDATE (Nov 2014):

Paul’s work has now been published in a paper in Biology Open (Biol Open. 2014 Nov 13. pii: BIO20149761. doi: 10.1242/bio.20149761). One of the important findings in his paper is that most of the cadhesome components were already found in pre-metazoan unicellular organisms. Those organisms also had cadherin, but it’s tail didn’t have the capability to bind catenins. So, the switch from unicellular to metazoa didn’t involve the “invention” of many new proteins, but rather a few mutations in the cytoplasmic tail of cadherin that would allow it to bind catenins. The figure below (taken from the paper) demonstrates the early evolutionary origin of the cadhesome.

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The Cadherin Adhesome [Completed Project]

Previously, we successfully constructed the integrin adhesome, which is a database of all known components of integrin mediated adhesion and the interactions between them. We have gone on to analyze the adhesome network using systems biology approaches. We performed a similar literature based construction of a network of adherens junction components, we named the Cadhesome. The first version of our literature-based cadhesome was published in JCS in January 2013.

We went on to use proximity ligation (BioID) followed by mass spectrometry to uncover many new proteins that associate with E-cadherin at cell-cell junctions (or independently of cell-cell adhesion). Moreover, by tagging identified proteins with GFP, we determined the subcellular localization of 83 putative E-cadherin–proximal proteins and identified 24 proteins that were previously uncharacterized as part of adherens junctions. The following figure depicts the resulting comprehensive E-cadherin interaction network of 79 published and 394 previously uncharacterized proteins. This work was published in Science Signaling 7(354), rs7.

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Ultrastructure of adherens junctions [completed project]

Despite three decades of intense research on cadherin biology we know shockingly little about the physical structure of adherens junction. How are the cadherins arranged within the membrane? how are they connected to the cytoskeleton? Are the adaptors and regulators organized in any particular way? The reason for our ignorance has to do with the limitation of our observation methods: there is a gap in our ability to visualize objects between the resolution of X-ray diffraction crystallography (~0.1nm) and the resolution of conventional light microscopes (~300nm). As it happens, the scale of protein complexes, which is what the adherens junction is made of, is in the size range of 30-100 nm – right smack in our blind spot! Electron microscopy has adequate resolution, but sample preparation of adherens junctions is a challenge and its difficult to label proteins so it is difficult to know what you are looking at in the electron densities.

Thankfully, in the past couple of years, a new method of microscopy was invented, which cleverly overcomes the diffraction limit of light and enables imaging immunolabeled cells down to a resolution of 10-30nm. This method, referred to as super resolution microscopy, depends on taking multiple (up to 20,000) separate images of the same sample, each time recording only a small subset of fluorescent proteins or antibody-dyes. When only a single molecule is emitting light it is possible to pinpoint its location at very high resolution. After processing of all the images, a composite image that contains all the light sources (proteins) at high resolution is presented.

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In close collaboration with Tony Kanchanawong, we used 3D-STORM and iPALM to decipher the ultrastructure of cadherins, actin and a number of important adaptor and signaling proteins at adherens junction in various stages of assembly and under varying conditions of tension. We are also testing the effect of different mutations affecting cadherin binding on the organization of cadherins and ultrastructure of the junction. We found that loosely organized clusters of approximately five E-cadherin molecules that form independently of cis or trans interactions, and which are delimited by the cortical F-actin meshwork, are the precursors of trans-ligated adhesive clusters that make up the adherens junction.

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This work was published in Developmental Cell 32(2):139-54. Yao2015compressed See also MBInsights: Defining adhesion clusters

We also collaborated with Pakorn (Tony) Kanchanawong‘s group on elucidating the 3D stratification of adherens junctions formed between MDCK or C2C12 cells and a cadherin-coated surface using super resolution microscopy. This work revealed a separation between the membrane proximal cadherin-catenin layer and an actin-rich compartment that is bridged by an interface zone containing vinculin. The study also elucidated the mechanism of vinculin activation at adherens junctions, which entails both mechanical tension AND tyrosine phosphorylation.

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This study was published in Nature Cell Biology doi: 10.1038/ncb3456.

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Optogenetic tool for photoactivating diaphanous related formins [completed project]

Our interest in diaphanous related formins, such as mDia, stems from observations that mDia activity at adherens junctions has an impact on actin dynamics and cadherin concentration. However, there are conflicting reports in the literature. A major difficulty in assessing the function of mDia at cell-cell junctions is the lack of a reagent to activate endogenous mDia in the cell. Current approaches usually entail over expression of truncated constructs that are constitutively active, but do not necessarily localize correctly. We sought to develop a tool that would allow us to active endogenous mDia on demand. To this end we turned to optogenetics. In collaboration with Pei Hsuan and Klaus Hahn (UNC-Chapel Hill, NC) we have developed a photoactivatable Dia Autoinhibitory Domain (DAD), based on the fusion of a light oxygen voltage (LOV) domain to the DAD domain of mDia1. Our experiments have shown this to be a powerful tool for the activation of diaphanous related formins. For example, we discovered that when formins are activated in fibroblasts, actin polymerization takes place all along existing stress fibers and surprisingly the level of myosin in the stress fibers did not change. This work has been published in Cytoskeleton (Hoboken). 2013 Jul;70(7):394-407. doi: 10.1002/cm.21115. The plasmids for PA-DAD as well as full length constitutively active mutants are available from Addgene.

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