John Kolega, Ph.D. [http://deptdirectory.med.buffalo.edu/profile/facultyprofile.asp?ht=dd&fid=0F70L4CN4]

Pathology And Anatomical Sciences
University at Buffalo School of Medicine and Biomedical Sciences
315 Farber Hall
Buffalo, NY
Phone: (716) 829-3527
Email: Kolega@buffalo.edu

The Kolega group seeks to understand the molecular mechanics of endothelial locomotion during wound healing and angiogenesis with a primary focus on the role of myosin II and cytoplasmic contractility.  Fluorescent analogs and fluorescence imaging techniques are used to examine the organization, dynamics and regulation of myosin II in living endothelial cells as they move in tissue-culture model systems.  The following issues are being addressed:

I.  Establishing the time and location of myosin II assembly in migrating cells.  Defining when and where myosin II-based contractile structures form as a cell moves will clarify how myosin II contributes to locomotion.  These experiments will also reveal the mechanism(s) by which these components are redistributed as cells travel.  This is accomplished by:

(a) examining fluorescent analogs of myosin II in detergent-permeabilized cell models, and

(b) using a ratiometric imaging method that compares the distributions of assembly-competent and assembly-incompetent fluorescent analogs coinjected into living cells to reveal how assembly changes over time.    

II. Understanding the intracellular regulation of myosin II assembly and behavior.  Phosphorylation is known to alter the assembly and motor activity of isolated myosin II, and is believed to play an important role in regulating cell structure and dynamics during locomotion.  We are testing the relationship between specific phosphorylations on myosin II and the behavior of myosin II in the cytoplasm by examining the distribution of phosphorylated myosins with phosphorylation-specific antibodies, by observing the behavior of myosins with mutated phosphorylation sites, and by tracking turnover of radio-labeled phosphate in migrating cells.

III. Defining the relationships between myosin II and locomotive activity in cells migrating in a three-dimensional matrix.  In vivo, endothelial movement rarely occurs on a flat substratum.  Therefore, we wish to examine endothelial behavior in a complex three-dimensional matrix.  An in vitro model of angiogenesis, in which endothelial cells invade a collagen gel, will be used.  Fluorescent analogs and optical sectioning of living cells as they migrate in collagen gels will reveal whether myosin II forms discrete contractile structures that define the location and polarity of particular movements.