We have used diverse fluorescence imaging approaches combined with quantitative analysis and mathematical modeling to investigate the characteristics of endomembrane organization in eukaryotic cells, including 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 unconventional organelles;and cytoskeletal and endomembrane crosstalk in polarized epithelial cells, hematopoietic niche 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 unconventional organelles, including autophagosomes, primary cilia and peroxisomes. 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. The peroxisome is involved in the oxidation of fatty acids, bile salts and cholesterol, and converts hydrogen peroxide to nontoxic forms. Using a photolabeling, pulse-chase strategy in living cells we demonstrated that peroxisomal membranes originate from the ER. To investigate the mechanism for peroxisome turnover, we attached monoubiquitin to peroxisomal membrane components facing the cytosol. We found this caused the peroxosome containing these components to be a substrate for autophagy: the peroxisome was engulfed by autophagosomal membranes and upon autophagosome-lysosome fusion, the peroxisome was degraded. The process was dependent on the ubiquitin-binding protein, p62. These results suggest that peroxisomal turnover occurs by autophagy through a pathway involving ubiquitination of peroxisomal membranes and p62-mediated autophagosome targeting. Hematopoietic stem/progenitor cells (HSPCs) reside in the bone marrow niche, where adhesive interactions with osteoblasts provide essential cues for their proliferation and survival. We used live cell imaging approaches to characterize both the site of contact between osteoblasts and hematopoietic progenitor cells (HPCs) and events at this site that result in downstream signaling responses important for niche maintenance. HPCs made prolonged contact with the osteoblast surface via a specialized membrane domain. At the contact site, portions of the specialized domain containing these molecules were taken up by the osteoblast and internalized into long-lived, SARA-positive, signaling endosomes. This caused the osteoblasts to downregulate Smad signaling and to increase their production of stromal-derived factor-1 (SDF-1), a chemokine responsible for HSPC homing to bone marrow. Targeted regulation of signaling and remodeling events within the osteoblastic niche microenvironment thus involves intercellular transfer- from HSPC to signaling endosomes within osteoblasts. Patterning in the Drosophila embryo requires local activation and dynamics of proteins in the plasma membrane (PM). How these events are coordinated before celullarization, in the absence of PM barriers, remains unclear. We used in vivo fluorescence imaging to characterize the organization and diffusional properties of the PM in embryos expressing different PM proteins. Before cellularization, the PM was polarized into discrete domains having epithelial-like characteristics. One domain resided above individual nuclei and has apical-like characteristics, while the other domain was lateral to nuclei and contains markers associated with basolateral membranes and junctions. Pulse-chase photoconversion experiments showed that molecules can diffuse in each domain but do not exchange between PM regions above adjacent nuclei. Drug-induced F-actin depolymerization disrupted the localization of PM polarity markers and abolished the restricted diffusion pattern in the PM. These findings suggest a new model of PM organization in the syncytial embryo, in which epithelial-like properties and an intact F-actin network compartmentalize the PM and shape morphogen gradients.
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