Projects Involving Cell Migration
Dissecting the mechanism of cell mechanotransduction.
One of the important questions of cell biophysics which need to be addressed is "how cells sense environment and more specifically, how mechanical signals (i.e. external and internal forces) are transformed into a complicated net of biochemical signaling." This problem is approached in a variety of ways; one of them is to measure changes in the forces exerted by cells in response to various environmental factors like substrate stiffness, specific drugs influencing cellular processes or quality/quantity of extracellular matrix. Our lab employs a sophisticated biophysical method of measuring the deformations of an elastic substrate in order to determine forces generated by cells attached to that substrate. That way we can observe the changes in cell tractions caused by certain, well-defined factors and relate them to downstream mechanochemical signals. This rotation will involve the following steps: (1) Learn the theoretical principles of the traction assay followed by the “hands on” experience of preparing 100 mm thick elastic substrates. (2) Learn the principles of high resolution optical microscopy – transmitted light and fluorescence modalities. (3) After becoming familiar with the experimental procedure, a set of specific inhibitors of Focal Adhesion Kinase (FAK) molecule will be used to investigate the role of the FAK in the generation of cell tension and maintenance of cell polarity.
Role of talin phosphorylation in cell migration.
One of our research interests is in how signals regulate cell motility. Currently we are focused on the role of cdk5-mediated phosphorylation of talin in cell migration. Cdk5 phosphorylates talin at Ser425 in vitro and in vivo. Expressing mutants in which the phosphorylation site has been changed to alanine or aspartic acid inhibits the migration of a human neuroblastma cells, indicating a role of talin phosphorylation in cell migration. Interestingly, talin is associated with Smurf1, an ubiquitin ligase. Phosphorylation of talin by cdk5 significantly inhibits smurf1 binding. Substitution of Ser425 with Aspartic acid also inhibits the binding to Smurf1. Moreover, expression of the ubiquitin ligase causes talin degradation. Our ongoing work is to understand the molecular mechanism by which cdk5-mediated talin phosphorylation regulates its degradation and how this degradation is involved in cell migration. Our hypothesis is that the association of Smurf1 to talin may target talin for degradation, resulting in integrin inactivation and focal adhesion disassembly, while cdk5-mediated phosphorylation of talin may prevent talin degradation, thus promoting focal adhesion formation. This rotation will involve aspects of testing this hypothesis.
Studying adhesion dynamics in rapidly moving cells.
Crawling cells need to adhere on substrates in order to generate the traction required for them to move. Cells have different kinds of adhesion structures, the most extensively studied of which are focal adhesions. “Close contacts” are another type of adhesion structure in which the membrane is about 20-50 nm from the substrate and more weakly attached. Close contacts have remained elusive owing to the fact that they do not form discrete, easily identifiable structures like focal adhesions and specific molecular markers for them are yet to be determined. However they are an important part of the adhesion machinery of rapidly moving cells such as keratocytes and possibly epithelial cancer cells that have been stimulated to become motile. Using the latter cell type as a model system, in this rotation, we will study the dynamics of close contacts using Interference Reflection Microscopy (IRM). We will correlate these with the dynamics of GFP labeled adhesion proteins such as Vinculin, alpha-Actinin, Paxillin etc. using TIRF (Total Internal Reflection Fluorescence) microscopy. These studies will advance our knowledge about how cell attachment and speed are regulated by this poorly understood, yet important, adhesion.
Mechanisms of cortical oscillations in cells.
Several years ago we observed that cortical oscillations could be induced in spreading cells by depolymerizing microtubules (Pletjuskina,et al. 2001. Cell Mot. & Cytoskeleton 48: 235). The oscillation was hypothesized to be caused by competing action of calcium (activation of contractility) and Rho (inhibition) pathways. Theoretical modeling suggested that no oscillations in active Rho concentration were required for oscillations to occur. To check this prediction, in this rotation, we will simultaneously measure and analyze the evolution of calcium, cortical behaviour, and Rho, the later using biosensors developed in Dr. Klaus Hahn’s laboratory (Dept. Pharmacology). The data will be analyzed and incorporated into a mathematical model. This project also has a computational aspect for those interested in mathematical modeling:
- Development and analysis of cortical oscillations in spreading cells using a set of ordinary differential equations describing biochemistry of contractility;
- Development and analysis of cortical oscillations in spreading cells using new graphical systems biology tool: CMAP (causal mapping) (Weinreb et al. 2006. Cell Mot. & Cytoskeleton 63:)
Studies involving Chromophore Assisted Laser Inactivation to selectively impair function of cytoskeletal regulatory proteins.
- Screen Capping Protein depleted cells for coordinated regulation of other actin regulatory proteins, particularly the actin bundling proteins and proteins involved in filopodia formation. If any of these are overexpressed when CP is knocked down, we will overexpress them in a CP knockdown/rescue cell line and repeat EGFP-CP CALI under these new conditions.
- Generate a PAK1 knockdown/rescue cell line. This will invlove cloning shRNAi for PAK1 and an RNAi resistant version of EGFP-PAK1 into a knockdown/rescue lentiviral vector, infecting cells, FACS sorting the cells, making single cell clones, and screening the clones for total knockdown of PAK1 and physiological rescue with EGFP-PAK1.
Projects Involving Membrane Microdomains and HIV Infection
Effect of amphipathic peptides on clusters of viral membrane-associated proteins.
VanCompernolle, et al (J Virol 79:11598) investigated the effect of several antimicrobial amphipathic peptides derived from amphibian skin on infectivity of HIV-1 virions. Preincubation of HIV-1 virions with micromolar quantities of caerin 1.1, caerin 1.9 and maculatin 1.1 caused a significant, dose-dependent decrease in viral infection of Dendritic Cells due to disruption of the viral envelope. This rotation project will determine whether these antimicrobial peptides might also influence the aggregation of viral proteins into highly clustered membrane distributions. During the process of influenza virion budding, HA forms discrete clusters on the surface of the cell membrane and lipid-protein interactions seem to be important for the formation of these HA clusters. This rotation project will determine whether amphipathic peptides disrupt the clustering of HA in host cells as assessed by fluorescence microscopic techniques.
Optical discrimination of viral protein distributions in planar and highly curved membrane geometries.
During the processes of enveloped virus budding or endocytosis, highly curved membrane structures are evolved. It would often be informative to be able to distinguish these different geometries based on fluorescence images of labeled viral membrane associated proteins. For example, the HIV-1 polyprotein Gag is necessary and sufficient for budding of virus-sized particles from the plasma membrane of a host cell. During this process Gag goes from a relatively planar patch of host membrane to a highly curved budding structure. Live cell studies of the detailed kinetics and cellular location of budding would benefit from an ability to distinguish these structures; however, the HIV-1 virion’s diameter is only ~120 nm. The z-axis difference between a planar distribution of viral protein and a budding distribution would be similar to the virion diameter meaning that these two types of structures would both fall within the same diffraction limited volume, thus making them indistinguishable on the basis of z-axis scanning. However, the concentration of viral protein along the z-axis extension of the budding virion should cause a brighter and more narrowly distributed signal from labeled viral protein in the diffraction limited volume of the bud and surrounding host membrane as compared to the same amount of protein in a planar geometry. The rotation project will determine whether the unbudded and budding states are distinguishable based on a detailed examination of the point-spread function (PSF) of spots of labeled viral protein.
Analysis of HIV-1 particles budding out of primary Dendritic Cells.
Human immunodeficiency virus type 1 (HIV-1) is a complex retrovirus that is highly dependent on cellular mechanisms for successful replication. This is particularly true with regard to the assembly and budding processes that occur during biogenesis. HIV-1 particle assembly and subsequent budding is a highly-ordered, multistep process promoted by the viral Gag precursor protein, and has been shown to be intimately linked with the cellular exocytic pathway. While these processes have been extensively described in primary T cells and macrophages, they have not been fully elucidated in primary dendritic cells (DC). As such, in this rotation, we will determine the site at which HIV-1 particle budding occurs in DC. To assess this, DC expressing HIV-1 Gag will be visualized at the ultrastructural level using immunoelectron microscopy. To further determine localization of budding particles, DC expressing GFP-labeled HIV-1 Gag will be examined using confocal microscopy, as well as total internal reflectance fluorescence (TIRF) microscopy.
Characterization of surface molecules on the cell membrane by single particle tracking of quantum dots.
This rotation project involves the technical development of employing quantum dots to track individual cell surface molecules. Quantum dots attached to specific surface molecules will be used to investigate the diffusion behavior of such molecules. The trajectory will be extracted from the movies recorded under the microscope and statistical methods will be used to analyze the trajectories. The diffusion rates, confinement proportions and other relevant parameters of different molecules will be determined.