Research Area I. Morphological Oscillations in Cells

Background: An important property of all cells is their ability to sense and respond to their environment. Mounting the appropriate response to an environmental challenge often involves large-scale changes in cell morphology. For example, environmental cues such as hormones or growth factors can lead to cell differentiation, proliferation, or migration. Key components of the cytoskeleton that mediate these responses are the actomyosin and microtubule systems. As a model system for investigating cytoskeletal dynamics, we focus on the morphological oscillations that occur after cell rounding. These oscillations can occur spontaneously but are highly enhanced by microtubules depolymerization.

Project I. Proteins involved in the cell oscillation phenotype

Using high resolution confocal imaging, we demonstrated that during the morphological oscillations the actin-myosin cortex and membrane are constantly reshaped and the cortex polarizes toward alternate sides of the cell. Furthermore using biochemical approaches we have shown that these morphological oscillations require the activity of the membrane bound signaling protein RhoA, which is involved in shaping the actin cortex. In turn, proteins associated with the actin cortex recruit negative regulators of RhoA. Thus the biochemical processes that regulate the oscillations are coupled to the biophysical mechanism that generates the active forces required for the oscillations. In this rotation, the student will ask what other actin binding proteins are involved in cortical remodeling during oscillations and what is the timing of their appearance in the cortical remodeling that is central to the oscillatory phenotype. We will express in a CHO cell line, which already has stable expression of EGFP-Lifeact, small peptide that binds to f-actin, or EGFP-Myosin Regulatory Light Chain, additional fluorescently tagged actin-binding  proteins of interest.  This project will involve state of the art imaging of living cells. Two color spinning disk confocal microscope measurements will be made with one channel imaging actin or myosin as a reference and the other channel imaging the actin-binding protein to be imaged. In collaboration with David Adalsteinsson, in the Department of  Mathematics at UNC, we will employ advanced of 3D reconstructions to permit the timing of various proteins involved in cortical remodeling.

Project II. Relationship of oscillating cells to cells undergoing division

We hypothesize that oscillating cells are similar, in some key ways, to cells undergoing cytokinesis and therefore employ similar molecular mechanisms and signaling pathways. If this is true, the morphological oscillations we observe would provide a useful model system for cell biologists to investigate mechanistic details of the cytoskeletal apparatus employed during cytokinesis. To explore the similarities between this ring structure and the contractile ring in cytokinesis,  we will investigate the distribution of several proteins, known to be specific for the contractile ring, in oscillating cells. These proteins include: Polo kinase, that promotes the accumulation of active RhoA-GTP in the cleavage furrow; CYK-4,  a Rho family GAP (GTPase activating protein), and kinesin-6, a microtubule motor, that are key proteins of centralspindlin complex; the Ect 2 RhoGEF (Guanine nucleotide Exchange Factor) that activate RhoA at the equatorial cortex; and Anillin, which is not only necessary for the organization and/or recruitment of structural components of the ring, but also for linking these components to signaling proteins that control cytokinesis and mitotic exit. This project will involve state of the art imaging of living cells. Two color spinning disk confocal microscope imaging will be employed and in collaboration with David Adalsteinsson, in the Department of  Mathematics at UNC, we will perform advanced of 3D reconstructions of images of living cells.

Project III. Systems biology investigation of cell oscillations

In general, these cell morphological oscillations provide insight into interactions between the actomyosin and microtubule systems on the global scale since depolymerizing microtubules dramatically changes the steady state of the cell system, throwing it into violent oscillations.  For this reason, this experimental system provides a test bed for systems biology approaches linking regulatory networks to mechanical behavior on the level of single cells.  This is a collaborative effort involving several groups at UNC. The general approach is to model the cell cortex as a liquid crystalline shell whose oscillations are driven by a forcing function that dictates where and when cortical contractility occurs.  The forcing function is hypothesized, with good grounds, to involve the Rho family of small GTPases whose function is modeled in a reaction-diffusion context by Prof. Tim Elston in the Department of Pharmacology at UNC.  Cortical shell dynamics are modeled by Prof. Greg Forest in the Department of Mathematics at UNC.  Rotation students will be involved in subprojects with Drs. Elston and Forest that link experiment to theory.

Research Area II. Cell traction analysis

Project I. Measuring cell tractions

This project will involve measuring the tractions exerted by living cells on a substrate as they migrate.  These tractions form an input to a theoretical description of the migration of simple-shaped cells that is being developed by our collaborators at UC-Davis. Our lab employs a sophisticated biophysical method to measure the deformations of an elastic substrate in order to determine traction forces generated by cells attached to that substrate. 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 micron thick elastic substrates. (2) Learn the principles of high-resolution optical microscopy in transmitted light and fluorescence modalities.

Project II. Keratocyte migration mechanism

Keratocytes are a type of fast moving cell that grows as a continuous sheet on fish scales. They can be maintained in culture by letting the whole cell sheet migrate from fish scale onto various substrates. Trypsin and calcium free PBS break down the cell-cell connections so that each individual keratocyte migrates on its own. When keratocytes are dissociated from each other they form a unique canoe shape and the leading edge is made of a large and flat fan-shaped lamellipodia. The keratocyte serves as a good model to study the function of lamellipodia in cell migration. In our studies we load keratocytes with caged-Thymosin β4, which can bind to actin monomer and inhibit actin filament growth after photoactivation. Previous studies done in our group had shown that photouncaging Thymosin β4 at wing area of keratocyte could change the direction of the migrating cell. The current project is focused on understanding the mechanism underlying the turning. We are tackling this question by using mathematical modeling and imaging methods. The work involves measuring the deformations of an elastic substrate under cells in order to determine traction forces generated by cells. The cell traction changes caused by Thymosin β4 can be used for testing a mathematical model for keratocyte migration.

Research Area III. Unusual membrane domains in dendritic cells containing C-type lectins

Background: C-type lectins are all-purpose pathogen receptors for dendritic cells, a key cell in the immune response.  C-type lectins, including DC-SIGN, are organized into domains on the plasma membrane and serve as receptors for pathogens ranging from HIV and the Ebola virus to yeasts.  We have found that the domains are composed of nanoscale subdomains are surprising stable in which the component C-type lectins under no lateral diffusion in the plane of membrane.  The factors stabilizing these important domains are a subject of intense interest.

Project 1: What motifs of the DC-SIGN molecule are important for domain stability?

In this rotation, the student will investigate which region/domain of DC-SIGN facilitates the membrane microdomain formation.  This will be accomplished by truncating each motif of DC-SIGN and investigating whether domains are formed and stable using fluorescence microscopy. Advanced fluorescence microscopic techniques will be employed to determine lateral movement in the plane of the membrane.  These methods include single particle tracking of quantum dots, line-scan fluorescence correlation spectroscopy, and fluorescence recovery after photobleaching.

Project 2: Identification of candidate molecular partners conferring stability to DC-SIGN membrane domains

In this project, we will first crosslink DC-SIGN and its potential partners in the domain by immunoprecipitating DC-SIGN to identify possible co-precipitating proteins by western-blot. Successful isolation of domain components will be a precursor to a full mass spectroscopy investigation followed by proteomic and lipidomic analysis   

Project 3: Reconstitution of protein microdomains on model-membranes:

This rotation project involves isolating and purifying DC-SIGN that is known to form membrane microdomains in vivo.  Following purification, these proteins will be introduced into model membranes and their distribution and dynamic properties will be studied to determine whether clustering is a self-assembly process.

Research Area IV.  Investigating the Carbohydrate ligands that drive fungipod responses

Background:  C-type lectins are carbohydrate-binding receptors used by innate immune cells to recognize molecular signatures of pathogens.  They are especially important for host defense against environmental and pathogenic fungi because the fungal cell surface is dominated by carbohydrate ligands such as mannoproteins, a class of cell wall proteins bearing highly branched N-linked polymannose glycans (“mannan”).  Mannoproteins of S. cerevisiae are highly stimulatory for a novel cellular protrusive organelle, the fungipod, which is formed by immature dendritic cells during fungal recognition by the C-type lectin CD206. 

Project 1: Mannans as the stimulators of fungipods

This rotation project will undertake to prove that mannan is the fungipod stimulatory part of S. cerevisiae mannoproteins through biochemical purification of mannan, assembly into particulate ligands and imaging of fungipod responses.  The project will then proceed in several possible complementary directions including: 1) chemical decomposition of mannan to determine what mannan substructures are responsible for fungipod induction, 2) genetic dissection of mannan structure using yeast mutants with altered mannan structure to understand what parts of mannan are required to induce fungipods and 3) extending similar work to the Candida species fungal pathogens and their mannans.

Project 2: Imaging early dendritic cell-yeast contact site formation

The fungipod evolves over ~1 hour of yeast attachment to a dendritic cell’s surface.  Furthermore, fungipods form efficiently to yeast-sized mannoprotein particles but only very poorly to smaller ligands (i.e., 1 um) bearing the same surface density of mannoprotein.  To understand how fungipods are formed and the size-discriminating aspects of this response, we must study the early dynamics of pre-fungipod contact site formation.  This rotation will address CD206 transport to and accumulation in the dendritic cell-yeast contact site to determine the mechanism and kinetics of contact site formation using live cell imaging and single particle tracking.  This rotation could also explore the use of fluorescent actin markers to investigate cytoskeletal dynamics leading up to the fungipod, a structure that is heavily dependent upon the actin cytoskeleton.

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