Date of Graduation
5-2015
Document Type
Dissertation (PhD)
Program Affiliation
Neuroscience
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
John O’Brien, PhD
Committee Member
Joseph L. Alcorn, PhD
Committee Member
Steve Massey, PhD
Committee Member
Christophe P. Ribelayga, PhD
Committee Member
Eric C. Swindell, PhD
Abstract
Gap junctions (GJ) formed of Cx36 show tremendous functional plasticity on several time scales. Changes in connexin phosphorylation modify coupling in minutes through an order of magnitude, but recent studies also imply involvement of connexin turnover in regulating cell-cell communication. We utilized Cx36 with an internal HaloTag to study Cx36 turnover and trafficking in cultured cells to discriminate newly formed and pre-existing Cx36. New Cx36 in cargo vesicles was added directly to existing gap junctions and newly made Cx36 was not confined to points of addition, but diffused throughout existing gap junctions. Existing connexins also diffused into photobleached areas with a half-time of less than 2 seconds. Recovery of connexin was impaired when laser power was focused and phototoxicity may be responsible. To better understand mechanisms of turnover we studied the role of cytoskeletal elements, actin filaments in particular, in Cx36 vesicle trafficking and GJ mobility. Phalloidin labeling showed that thick actin bundles connected all edges of GJ plaques, but actin filaments were rare within. Actin filaments were found associated with small, chase-labeled delivery vesicles. Many GJs showed substantial numbers of finger-like filadendrites extending from both the edges and the center of the plaques, and the morphology of these filadendrites changed at a fast pace. Double labeling of HaloTag ligand and phalloidin showed that these filadendrites colocalized with thin actin filaments. Disruption of actin filaments with Cytochalasin D caused loss of GJ at cell-cell contacts. Treatment with Latrunculin A, which prevents new actin elongation, did not disrupt GJ vi plaques and only partially suppressed Cx36 turnover, but eliminated the filadendrite extensions. In conclusion, studies of Cx36-HaloTag revealed novel features of connexin trafficking and demonstrated that phosphorylation-based changes in coupling occur on a different time scale than turnover. The role of rapid mobility of elements of GJ plaques in functional plasticity is unknown, but we hypothesize that it may relate to the mechanisms that control turnover of connexin protein.
Keywords
Connexin, traffikcing, turnover, plasticity, fluorescent imaging, Gap Junction, Cx36