Title

Chemotaxis and phagocytosis: Two distinct mechanisms of polarized cytoskeletal reorganization

Date of Completion

January 2003

Keywords

Biology, Cell

Degree

Ph.D.

Abstract

Chemotaxis and phagocytosis are two distinct mechanisms that occur as a result of extracellular activation, inducing polarized cytoskeletal reorganization. Phagocytosis is defined as binding and internalization of a particle greater than 0.5 μm in diameter. The binding induces the propagation of a trans-membrane signal that is relayed to the cytosolic region of the cell. This signal results in localized actin polymerization at the site of internalization. Here we report the first attempt at introducing a live, viable substrate (bacteria) expressing a red fluorescent protein as a marker that allows for the plate fluorometric and flow cytometric determination of uptake and degradation rates in D. discoideum. We now have an assay that allows for the reproducible quantification of these rates as well as image analysis of the localized molecular mechanisms involved. ^ Chemotaxis is the process whereby recognition of an external gradient induces polarized actin polymerization resulting in persistent and directional cell body translocation. Binding of chemoattractant ligand activates a signal transduction cascade that promotes the formation of a pseudopod at the “front” of the cell and subsequent retraction of the midbody anteriorly, resulting in “forward” cell motility. The development of an under-agarose folate chemotaxis assay in which cells must (a) recognize a chemoattractant gradient, and (b) deform the agarose upwards while flattening themselves as they transition under the agarose, allows us to analyze this process. We have found that in the restricted environment of this under-agarose assay, cells lacking the motor function of myosin II are able to chemotax normally, whereas cells proposed to be lacking the cross-linking function of myosin II are severely limited in their motile characteristics. These results have been determined via computerized measurement of cell motility and image analysis of GFP fusion proteins. We hypothesize the cross-linking function of myosin II is critical for cell motility in a restrictive environment. ^ Phagocytosis and chemotaxis are specialized mechanisms of cellular polarity. I propose the hypothesis that both of these mechanisms are types of polarized cytoskeletal reorganization that are the result of the induction of specific types of receptors. ^