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 recently 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.
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.
Investigating the interplay between Actin Polymerization and Cadherin-mediated cell-cell adhesion in mammalian epithelial cells
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 attempt to dissect the role of actin polymerization mediated by formins in regulating E-Cadherin clustering and dynamics at AJs. Furthermore, we are also interested in understanding the interplay between different classes of nucleators (such as formins and the Arp2/3 complex) and their contributions to actin polymerization at apical and lateral junctions.
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. Plastin/fimbrin is an evolutionary-conserved actin-bundling protein with two tandem repeats of calponin homology (CH) domains. To examine the role of C. elegans plastin, PLST-1, in early embryogenesis, we are characterizing the plst-1(tm4255) allele, a deletion that abrogates the third and fourth CH domains.
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.
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, and are in the process of analyzing it and comparing it to the integrin adhesome. The first version of our literature-based cadhesome was published in JCS in January 2013.
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.
In close collaboration with Tony Kanchanawong, we are using 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.
Now we are working on the ultrastructure of a number of important adaptor and signaling proteins at adherens junctions.
It is well established that integrin-mediated adhesions (such as focal adhesions) are mechano-responsive: they grow when force is applied to them and they disassemble when tension is relieved. Stretching of cas and talin, exposing vinculin binding sites, recruitment of LIM domain proteins and tyrosine dephosphorylation of paxillin have all been implicated in integrin-mediated mechanosensing.
In recent years it is becoming apparent that adherens junctions are also sensitive to the level of tension at the junction. Inhibition of tension leads to disassembly of adherens junctions (albeit at a slower rate compared to focal adhesions), and increasing contractility within cells (by activating RhoA, for example) leads to reinforement of the junctions. The one mechanism proposed to date for junctional mechanosensing is the stretching of alpha-catenin by force, exposing a binding site for vinculin. We are working towards identifying novel components responsible for force-induced junction assembly by two methods: 1. An siRNA screen of candidate genes applied to a robust assay for force-induced assembly. 2. Differential mass spectroscopy of isolated cadherin adhesions under high and low tension conditions.
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. Ongoing work is aimed at using this tool for the study of formins at adherens junctions. The plasmids for PA-DAD as well as full length constitutively active mutants are available from Addgene.
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.
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.
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.