We have used diverse fluorescence imaging approaches combined with quantitative analysis to investigate the characteristics of endomembrane organization and dynamics in eukaryotic cells and whole organisms. Among the areas being investigated are: mitochondria, autophagy, secretory vesicles, primary cilia, cytokinetic membrane abscission and membrane furrowing. Mitochondria are highly dynamic organelles that mediate essential cell functions such as apoptosis and cell-cycle control in addition to their role as efficient ATP generators. It is known that mitochondrial morphology changes are tightly regulated, and their shape can shift between small, fragmented units and larger networks of elongated mitochondria. Using live cell imaging, we observed that mitochondrial elements become significantly elongated and interconnected shortly after nutrient depletion. This mitochondrial morphological shift depended on the type of starvation, with an additive effect observed when multiple nutrients were depleted simultaneously. The starvation-induced mitochondrial elongation was shown to occur by downregulation of dynamin-related protein 1 (Drp1) through modulation of two Drp1 phosphorylation sites, leading to unopposed mitochondrial fusion. We further demonstrated that mitochondrial tubulation upon nutrient deprivation protected mitochondria from autophagosomal degradation. This could permit mitochondria to maximize energy production and supply autophagosomal membranes during starvation. We demonstrated a regulatory link between mitochondrial fission activity and cell cycle exit as the follicle cell layer develops during Drosophila melanogaster oogenesis. This was accomplished by performing live-cell imaging after manipulation of key mitochondrial fission/ fusion proteins. We observed that posterior-localized clonal cells in the follicle cell layer of developing ovarioles with down-regulated expression of the major mitochondrial fission protein DRP1 did not exit the cell cycle. Instead, they excessively proliferated, failed to activate Notch for differentiation, and exhibited downstream developmental defects. Reintroduction of mitochondrial fission activity or inhibition of the mitochondrial fusion protein Marf-1 in DRP1-null clones reversed the block in Notch-dependent differentiation. When DRP1-driven mitochondrial fission activity was unopposed by fusion activity in Marf-1depleted clones, premature cell differentiation of follicle cells occurred in mitotic stages. Our findings thus revealed that DRP1-dependent mitochondrial fission activity regulates the onset of follicle cell differentiation during Drosophila oogenesis. The results link, for the first time, mitochondrial dynamics and cell fate determination. The physical separation of two daughter cells at the end of mitosis, known as cytokinetic abscission, involves cleavage of a narrow, microtubule-based, intercellular bridge that connectes two nascent daughter cells arising during cell division. We have been investigating the role of the endosomal sorting complex required for transport (ESCRT)-III complex in this process. Using high resolution, quantitative imaging of ESCRT-III during cytokinetic abscission, we observed that ESCRT-III initially assembles at the midbody dark zone and then polymerizes outward to the site of cytokinetic abscission. Integrating these observations with the known biophysical properties of ESCRT-III complexes, we then formulated and tested a computational model for ESCRT-mediated cytokinetic abscission. In this model, ESCRT-III forms a fission complex that drives constriction and abscission of the intercellular cytokinetic bridge. The ESCRT-III fission complex arises as a result of VPS4-enabled breakage of the initial ESCRT-III oligomer, polymerizing at the edge of the midbody dark zone. Once formed, the fission complex constricts to its spontaneous diameter of 50 nm, while sliding along the intercellular bridge away from the midbody dark zone. Sliding continues until the fission complex reaches the minimal elastic energy of the bridge membrane, which is where fission occurs. We substantiated this model by theoretical analysis of the membrane elastic energy and by experimental verification of the major model assumptions. Currently, we are exploring whether ESCRT-III polymerization coupled with breakage and sliding of a constricting membrane-bound ESCRT-III complex to a location corresponding to minimal elastic energy of the membrane is a conserved property of other ESCRT-mediated processes (i.e., human immunodeficiency virus budding). Rab proteins are important regulators of insulin stimulated GLUT4 translocation to the plasma membrane (PM), but the precise steps in GLUT4 trafficking modulated by particular Rab proteins remain unclear. To clarify this issue, we used TIRF microscopy to monitor GLUT4 trafficking under basal conditions and during insulin signaling, identifying Rab proteins associated with GLUT4 storage vesicles (GSVs) and their mode of action on GLUT4 trafficking. Screening a large set of Rab proteins for colocalization with GLUT4 for association with IRAP-pHluorin vesicles fusing at the PM in response to insulin stimulation, and for PM recruitment under insulin stimulation, we discovered that only Rab10 associates with GSVs and mediates GSV peripheral translocation after becoming activated. The Rab GTPase activation protein, AS160, negatively regulated Rab10 activity on GSVs. Furthermore, we found that Rab10 interacted with the actin motor protein myosin-Va. This facilitated the translocation of GSVs to sites at the PM where they could fuse. High expression levels of a short tail form of myosin-Va lacking the actin-binding domain decreased GLUT4 delivery to the PM. Thus, even though multiple Rab proteins regulate the trafficking of GLUT4, it is only Rab10 in coordination with myosin-Va that mediates the final steps of GSV translocation to the PM under insulin stimulation. Primary cilia have major roles in sensing and transmitting information into cells. We hypothesized that like motile cilia, primary cilia have the potential to form adhesions. To test this, we examined cilia in two different tissues: photoreceptors in the retina and cholangiocytes in liver. In both of these environments we observed cilia form contacts with each other. Using a cell culture model combined with fluorescent cell imaging we demonstrated that cilia from nearby cells could form persistent, regulated, glycoprotein dependent, cilia-cilia adhesions. In addition, we found evidence for cellular control of adhesion release. We suggest that like the contacts made by motile cilia, adhesion of primary cilia is functionally relevant. These results also suggest that mammalian primary cilia may be more than passive, solitary receivers. The effects of the geometry of the early syncytial Drosophila embryo on the effective diffusivity of cytoplasmic proteins are poorly understood. Using a combination of mathematical/computational modeling and live-cell imaging, we characterized the effects of dynamic syncytial geometry on the effective diffusivity of cytoplasmic proteins in the Drosophila syncytial blastoderm. We found that the presence of transient mitotic membrane furrows results in a multiscale diffusion effect that has a significant impact on effective diffusion rates across the embryo. Based on these results, we proposed that one role of syncytial membrane furrows is to temporally regulate bulk embryonic diffusion rates to balance the multiscale effect of interphase nuclei, which ultimately stabilizes the shapes of various morphogen gradients.

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Waxse, Bennett J; Sengupta, Prabuddha; Hesketh, Geoffrey G et al. (2017) Myosin VI facilitates connexin 43 gap junction accretion. J Cell Sci 130:827-840
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