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 microtubule depolymerization.

Project I. Proteins involved in the cell oscillation (periodic protrusive) 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 during 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. We will employ advanced 3D reconstructions to visualize 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. We will employ advanced 3D reconstructions to visualize the timing of various proteins involved in cortical remodeling.

Project III.  Are periodically protruding cells a model of amoeboid migration?

Oscillatory phenotypes are frequently seen in cell and tissue biology. Yet except for the case of cardiomyocytes, the mechanisms that drive these oscillations are largely unknown. In early stage fly embryos, cells of epithelial origin exhibit oscillations in the actinmyosin network that are required for embryonic elongation and for apical constriction. The oscillatory nature of amoeboid type of motility is becoming more widely accepted.  The principle components of amoeboid movement include a simple polarized shape, dynamic pseudopodial protrusion and retraction, and flexible and oscillatory shape changes. Examples of amoeboid migration range from the movements of individual cells of the lymphoid system to the migration of metastatic cancer cells. However, studying amoeboid motion in molecular detail has proven challenging due to the difficulty of imaging cells moving in 3 dimensions. This obstacle is removed in our oscillating cell system.

Our analysis indicates important parallels between oscillating cell motility and blebby amoeboid migration, which is one principal mode of movement for metastatic cancer cells. Similar to the oscillatory phenotype, cells undergoing this form of locomotion lack focal adhesions and stress fibers that pull on the substrate to generate movement, but rather develop protrusive bulges that push through interstices in the extracellular matrix.  Consistent with amoeboid migration, confocal imaging of the actomyosin cortex during morphological oscillations indicates dynamic polarization of f-actin and myosin at the site of contractility. In amoeboid movement, the cell migrates through a series of aperiodic bulges. The protrusions exhibited by oscillating cells are similar to those seen during 3 dimensional amoeboid migration. Additionally, we have observed that oscillating cells can translocate during oscillatory motility, suggesting that these cells are capable of amoeboid-like migration. Also, similar to the oscillatory phenomenon, amoeboid movement is known to require high activity of RhoA, suppression of Rac activity, and involve phosphoinositides. We assume that because the oscillatory phenotype recapitulates a significant number of the amoeboid movement elements it could represent a good model for investigating the mechanisms that drive this important mode of migration, which has not received nearly as much attention as mesenchymal (fibroblastic) movement. Thus, in this project, we will further investigate and validate the possibility to use morphological oscillations as a biological model for 3D amoeboid migration.

Project IV. 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 and 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. Unusual C-type lectins  membrane domains in dendritic cells

Background: C-type lectins are all-purpose pathogen receptors expressed on the surface of dendritic cells, a key cell in human immune system.  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 Dengue virus to yeasts.  We have found that the domains are composed of nanoscale subdomains and that these domains are surprisingly stable, i.e., the component C-type lectins exhibit no lateral diffusion in the plane of membrane.  The factors stabilizing these important domains are a subject of intense interest.

Project 1: 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; this will be followed by 2D gel electrophoresis.  Spots on the 2D gel will be subjected to mass spec proteomics analysis.

Project 2: 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 III.  Investigating the interaction of Dengue virus with DC-SIGN domains

Background:   Dengue virus (DV), a small (~50nm) enveloped flavivirus, is a large and growing global health threat.  It is carried by mosquitoes and infects an estimated 50 to 100 million people each year and nearly half the world’s population is now at risk. Severe infections in certain individuals cause Dengue hemorrhagic fever, a life threatening condition and DV infections cause more than 20,000 deaths per year. DV uses DC-SIGN, as a primary receptor for entry into dendritic cells. Understanding the immunopathology of DV infection is crucial to development of safe vaccines and therapeutics.  This work is a collaboration between our laboratory and that of Prof. Aravinda de Silva in the Department of Microbiology and Immunology.

Project 1: Measuring the binding of mature and immature forms of DV to dendritic and cultured cells

This rotation project will develop imaging cytometry and flow cytometry assays to measure the binding of mature and immature forms of DV to both dendritic cells and other cultured cells. The project will involve fluorescent labeling of DV and fluorescence microscopic imaging and flow cytometry.

Project 2: Super resolution imaging of the binding of DV to dendritic and other cultured cells (planned)

In this rototation project, we will employ one of the super resolution techniques of PALM, STORM and Blink in the UNC-Olympus Imaging Research Center to study the interaction of DV with DC-SIGN receptors.  By employing the super resolution imaging technique, we hope to answer the questions: How many DC-SIGNs are required to stably bind mature and immature DV? Are the DC-SIGN nanodomains re-arranged in order to optimally bind pathogen?

Top  Home