We have used diverse fluorescence imaging approaches combined with quantitative analysis to investigate the characteristics of endomembrane organization in eukaryotic cells, including nonpolarized and polarized cell monolayers in tissue culture and in living embryos. Among the areas being investigated are: mitochondrial morphology and its regulation of cell cycle progression;the origin and dynamics of organelles;and cytoskeletal and endomembrane crosstalk in polarized epithelial cells and developing Drosophila syncitial blastoderm embryos. Related to our work characterizing mitochondria, we carried out live cell imaging experiments in cells stably expressing RFP targeted to the mitochondrial matrix to determine if there are changes in mitochondrial dynamism and function at different stages of the cell cycle. We found that mitochondria exhibit distinct morphological and physiological states at different stages of the cell cycle. In mitosis, mitochondria fragmented into hundreds of small units for partitioning into daughter cells at cytokinesis. Strikingly, at G1/S, mitochondria fused together into a single huge, dynamic filamentous system, unlike at any other cell cycle stage. Photobleaching of an area across this filamentous system revealed the mitochondrial matrix was continuous. The mitochondrial network also was electrically coupled and had a higher membrane potential than mitochondria at all other stages of the cell cycle. When the filamentous network or its membrane potential was disrupted, or its dynamics perturbed, cell cycle progression from G1 into S was arrested in a p53-dependent manner. Moreover, p21-overexpression, which induces a G1/S arrest, resulted in filamentous mitochondria with reduced matrix continuity and loss of electrical coupling. The data thus revealed that mitochondria dynamism and morphology undergo critical changes during the cell cycle that are sensed by the cell at G1/S to control cell cycle progression. We explored the origin and dynamics of organelles. One of these is the autophagosome. Autophagosomes form during autophagy, a highly conserved, bulk degradation pathway that is also involved in turnover of large aggregates and organelles within cells. In the initial step of this pathway, an isolation membrane forms in the cytoplasm through the activation of specific autophagy effectors. The membrane wraps around the protein aggregate or organelle to form a double membrane-bounded structure called the autophagosome. The autophagosome then targets to and fuses with the lysosome where the sequestered materials are degraded by various hydrolytic enzymes and recycled as amino acids for macromolecule synthesis and energy production. While emerging results have revealed the importance of autophagy in various biological and pathological processes, such as cellular remodeling, tumorogenesis and neurodegeneration, how this pathway operates is far from clear. We utilized various live cell imaging and molecular genetic approaches to investigate the membrane origin of autophagosomes and the signals that recruit substrates to this organelle. Our data revealed that the outer membrane of mitochondria serves as the membrane source during starvation-induced autophagy formation and maturation. Furthermore, ubiquitin modification acts as a targeting signal for delivery of small cytosolic proteins as well as larger organelles to autophagosomes. The primary cilium is a chemosensory and mechanosensory organelle. The axoneme of a cilium is composed of nine doublets of microtubules that extend out from the triplet microtubules of the centrosome. Primary cilia have been described as antennae because they often project away from the cell surface and they are able to receive signals (both chemical and mechanical) from the extracellular environment. Imaging this important organelle is essential to developing a better understanding of how it functions. By imaging live polarized epithelial cells we found that cilia can make direct contact with cilia of adjacent or nearby cells. Imaging live cells expressing different cilia-localized fluorophores revealed that the bridges are composed of cilia from individual cells: they are not a continuation from one cell to the next. Cilia bridges can be stable over many hours. Trypsin and other protein disrupting treatments have not disrupted the cilia-cilia adhesion. These findings suggest that cilia may do more than just passively sense the environment. They may be able to initiate and mediate cell-cell communication through direct contact. Dynamin is a large GTPase that assembles into scaffolds on membranes for membrane fission, reshaping and interactions with the cytoskeleton. We discovered a role of dynamin 2 (Dyn2) in regulating the acto-myosin contractile system of polarized epithelial cells. This actin regulatory role of Dyn2 provides a new framework for understanding the regulation of acto-myosin contractility involved in epithelial biogenesis and morphogenesis. We found that Dyn2 localizes to junctional membranes of polarized cells. Its knockdown blocks monolayer formation and disrupts tight junctional integrity in pre-existing monolayers. Its overexpression in a non-GTP-bound form (i.e., Dyn2 K44A) causes monolayers to apically constrict. The apical constriction phenotype depends on deacetylated cortactin, myosin II and ROCK activity and is associated with enrichment and stabilization of non-GTP-bound Dyn2 on apical junctional membranes. Conditions and/or mutants that shift dynamin to be in a predominantly GTP-bound state do not cause apical constriction. Hence, the nucleotide status of Dyn2 in membrane scaffolds appears to promote distinct actin-based activities in epithelial cells, with scaffolds enriched in non-GTP-bound Dyn2 regulating actin contraction at the apical junctional region in association with deacetylated cortactin.
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