The involvement of calcium signaling in cell polarity

Ling Huang, Purdue University

Abstract

My research projects address the mechanisms for establishing and maintaining cell polarity, with a focus on the involvement of intracellular Ca2+. Cell polarity refers to the existence of spatial differences in cells, such as asymmetric protein localization, which may lead to differing fates of sibling cells, directional cell migration or directional growth. I investigated the directional migration of zebrafish keratocytes and chick Schwann cells in response to applied electric fields (EFs), which have implications for wound healing and regeneration in vertebrates. I also studied the asymmetric divisions of Drosophila neuroblasts, which provides information for understanding how two cells with very different fates can arise from an initially spherically symmetrical cell. Many growing and motile cells respond directionally to small DC electrical fields (EFs). The mechanism of the response is not known, but changes in intracellular Ca2+ are widely assumed to be involved. We have used zebrafish keratocytes in an effort to understand the nature of the EF-cell interaction. We find that the adult zebrafish integument drives substantial currents outward through wounds produced by scale removal, establishing that keratocytes near the wound will experience endogenous EFs. Isolated keratocytes in culture turn toward the cathode in fields as small as 7 mV mm-1, and the response is independent of cell size. Epidermal sheets are similarly sensitive. The frequency of intracellular Ca2+ spikes and basal Ca 2+ levels were increased by EFs, but the spikes were not a necessary aspect of migration or EF response. Two-photon imaging failed to detect a pattern of gradients of Ca2+ across the lamellipodia during normal or EF-induced turning but did detect a sharp, stable Ca2+ gradient at the junction of the lamellipodium and the cell body. We conclude that gradients of Ca2+ within the lamellipodium are not required for the EF response. Immunostaining revealed a gradient of integrin β1 during EF-induced turning, and interference with integrin function attenuated the EF response. Neither electrophoretic redistribution of membrane proteins nor asymmetric perturbations of the membrane potential appear to be involved in the EF response, and we propose a new model in which hydrodynamic forces generated by electro-osmotic water flow mediate EF-cell interactions via effects on focal adhesions. According to this model, the shear forces generated by water flow are resisted primarily by focal adhesions on the anodal side, which causes an increase in focal adhesions there. As a result, the speed of migration of the anode-facing portion of the lamellipodium becomes greater than that of the cathode-facing portion, causing the keratocyte to pivot toward the cathode in the manner that is experimentally observed. This model for galvanotaxis is currently the only one that is consistent with the data. During Drosophila embryonic development, neuroblasts delaminate from epithelial cells and divide asymmetrically, and the neuroblasts then reorganize and undergo repeated asymmetrical divisions. Each asymmetric division produces a large new neuroblast and a smaller ganglion mother cell that generates neurons or glial cells for larval central nervous systems. We used the ratiometric calcium indicator fura-2 to study the temporal and spatial distributions of intracellular Ca2+ during neuroblast cell cycle. We also applied pharmacological reagents to perturb calcium distributions and inhibited Ca 2+/calmodulin dependent kinase II (CaMKII). We observed calcium gradients preferentially occurred at the apical portion of neuroblasts and gradual cytosolic Ca2+ increases during cell divisions. We discovered that normal calcium dynamics and activity of CaMKII were required for spindle pole formation and protein localization in the cell cortex. Schwann cells are derivatives of neural crest cells. Transplanted Schwann cells promote axonal regeneration in the peripheral nervous system, but the cues for guiding their migration are not well known. We have investigated the response of Schwann cells, cultured from the peripheral nerves of E7/8 chick embryos, to applied electrical fields. We found that they responded by migrating to the anode, and showed a significant anodal bias in directionality at 3 mV mm-1. This is the smallest electrical field that has been shown to affect cellular movement or growth in culture, and the anodal direction is surprising given the known cathodal responses of neural crest cells. The effective fields are considerably smaller than endogenous electrical fields that have been measured in embryonic tissues.

Degree

Ph.D.

Advisors

Robinson, Purdue University.

Subject Area

Neurosciences|Cellular biology

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