Research

Genetic Analysis of Chemotaxis in Eucaryotic Cells

Chemotaxis and phagocytosis are an integral part of immune response and play a key role in wound healing, angiogenesis, and embryogenesis. These fundamental cellular processes are found in all eukaryotes and have remained essentially unchanged throughout evolution. Research in the last fifteen years in D. discoideum showed that chemoattractants are sensed by the same basic signal transduction mechanisms as are many hormones, neurotransmitters, odorants. The receptors for these cell-cell signaling molecules activate heterotrimeric G-proteins that regulate phosphodiesterases, phospholipases, ion channels, and adenylyl cyclases. Our strategy is to exploit the genetic advantages of D. discoideum to discover mechanisms of sensing chemoattractant gradients and to apply this information to higher eukaryotic cells. 

We have identified many of the genes involved for these processes. There are four cell surface cAMP receptors, eight G-proteins α-subunits, unique β- and γ-subunits, and an adenylyl cyclase. Targeted gene disruptions have shown that many of these genes are essential for development and play central roles in chemotaxis, cell-cell signaling, and agonist-induced gene expression. Complementation of these null cell lines with randomly mutagenized libraries has allowed us to identify point mutations that alter specific functions of the receptors, G-protein subunits, or adenylyl cyclase. Current work is focused on biophysical studies of these mutant proteins. Complementation also provides a simple genetic test of the function of GFP-tagged proteins. With this approach, we are studying the dynamic distribution of the receptors, G-protein subunits, adaptors, and effectors in single living cells during chemotaxis and persistent stimulation. 

Consideration of the features of a chemotactic response presents several fascinating and unique challenges.  Shallow external gradients must generate sharply localized internal responses at the leading edges of the cells.  Moreover, cells at different points in the gradient sense equally well so there is a powerful mechanism for background subtraction or adaptation.  We have suggested that a balance between local excitatory and global inhibitory processes controls the response to chemoattractants.  An extensive series of studies in the last several years have indicated that the upstream components and reactions in the signaling pathway are uniformly localized in cells exposed to a chemoattractant gradient.  However, downstream responses such as PI (3,4,5)P₃ accumulation and actin polymerization are sharply localized towards the high side of the gradient, suggesting that these responses are selectively activated at the cell’s leading edge.  We have recently found that uniform stimuli transiently recruit and activate PI3Ks while PTEN is released from the plasma membrane.  Although chemoattractant receptors and G-proteins are evenly distributed along the cell surface, gradients of chemoattractant cause PI3Ks and PTEN to bind to the membrane at the front and the back of the cell, respectively.  This reciprocal regulation provides robust control of PI(3,4,5)P₃ and leads to its sharp accumulation at the anterior.  Interference with PI3Ks modifies chemotaxis while disruption of PTEN broadens PI localization and actin polymerization in parallel.  Thus, counteracting signals from the upstream elements of the pathway converge to regulate the key enzymes of PI metabolism, localize these lipids, and direct pseudopod formation. 

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This page was last edited 12/12/2003