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: (1) membrane partitioning and its role in protein sorting and transport in the Golgi apparatus, (2) biogenesis and dynamics of unconventional organelles, (3) mitochondrial morphology and its regulation of cell cycle progression, and (4) cytoskeletal and endomembrane crosstalk in polarized epithelial cells, hematopoietic niche cells, and developing Drosophila syncitial blastoderm embryos.&#8232;[unreadable] [unreadable] Included in our major findings is the characterization of secretory cargo transport through the Golgi apparatus. This important organelle processes and filters newly synthesized protein and lipid moving through the secretory pathway and is typically composed of 6-8 flattened cisternae arranged as a stack with surrounding vesicles and tubules. The most widely accepted model for intra-Golgi transport is cisternal progression. It postulates that the stack of cisternae seen in the Golgi apparatus constitute a historical record of progression from entry at the cis face to exit at the trans face. Recently arrived cargo molecules are confined in the cis-most cisterna, undergo initial processing there, and await the arrival of enzymes delivered by retrograde vesicles from more distal cisternae for subsequent processing. Cargo molecules remain within a given cisterna as it passes, conveyor-belt-like, through an average of seven locations within the Golgi stack on its way to the trans face and exit from the Golgi through transport carriers. A key prediction of this model is that newly arrived cargo exhibits a discrete lag or transit time before being exported. To test this prediction, we analyzed different classes of cargo molecules within the Golgi stack after they were fluorescently pulse-labeled and visualized quantitatively as they transited from the Golgi. Our results revealed there was an exponential loss of cargo from the entire Golgi rather than the linear pattern predicted by the classical model. Moreover, when transmembrane cargo entered the Golgi apparatus, it differentially partitioned between two different membrane environments- processing domains enriched in Golgi enzymes and export domains capable of budding transport intermediates. Based on results from these experiments, we constructed and tested a new model of intra-Golgi trafficking in which cargo molecules continuously partition between processing and export domains defined by different lipid compositions as they move up and down the Golgi stack. Cargo can be exported to the plasma membrane from within the export domain found within every cisterna. Simulation and experimental testing of this rapid partitioning model produced all the key characteristics of the Golgi apparatus, including polarized lipid and protein gradients, exponential cargo export kinetics and cargo waves. It thus represented a viable description of the mechanism of intra-Golgi transport. [unreadable] [unreadable] 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.