The Role of Actin Cytoskeleton in Endocytosis and Exocytosis in the Salivary Glands of Live Rodents

In the last two decades, mammalian cell biology has greatly benefited from major technological advances in light microscopy that have enabled imaging virtually any cellular process at different levels of resolution. However, mammalian cell biology has been studied primarily by using in vitro models. Cell culture models have been used the most, since it offers several advantages such as, being amenable to both pharmacological and genetic manipulations, reproducibility, and relatively low costs. However, their major limitation is that the architecture and physiology of cells in vitro differ considerably from the in vivo environment. This issue can be overcome by the use of intravital microscopy, which encompasses various optical microscopy techniques aimed at visualizing biological processes in live animals. Recent developments in non-linear optical microscopy resulted in an enormous increase of in vivo studies, which have addressed key biological questions in fields such as neurobiology, immunology and tumor biology. However, the motion artifacts derived from heartbeat and respiration have prevented the imaging of intracellular structures and limited the use of intravital microscopy to the analysis of tissue architecture or single cell behavior. In this respect, the goals of my thesis have been: 1) the development of an experimental system that enables visualizing subcellular organelles in live rodents for extended periods of time, and 2) the investigation of the role of the actin cytoskeleton in endocytosis and exocytosis in live rodents. Here, I describe the establishment of a model for studying endocytosis end exocytosis in the salivary glands of live rats and mice. Moreover, I show that both processes can be imaged in live animals and that their behavior and kinetics differ from what has been reported in in vitro systems. Next, I show that the salivary glands can be genetically and pharmacologically manipulated in situ, thus opening the door for the investigation of the molecular machinery regulating membrane trafficking in live animals. Finally, in the last part of my dissertation, I focus on specific approaches developed to study the kinetics of exocytosis of single secretory granules and discuss how the actin cytoskeleton plays a fundamental role in controlling this process.Doctor of Philosoph

Regulated Exocytosis: Novel Insights from Intravital Microscopy: Exocytosis and Intravital Microscopy

Regulated exocytosis is a fundamental process that every secretory cell uses to deliver molecules to the cell surface and the extracellular space by virtue of membranous carriers. This process has been extensively studied using various approaches such as biochemistry, electro-physiology, and electron microscopy. However, recent developments in time-lapse light microscopy have made possible imaging individual exocytic events hence advancing our understanding of this process at a molecular level. In this review, we focus on intravital microscopy a light microscopy-based approach that enables imaging subcellular structures in live animals, and discuss its recent application to study regulated exocytosis. Intravital microscopy has revealed differences in regulation and modality of regulated exocytosis between in vitro and in vivo model systems, unraveled novel aspects of this process that can be appreciated only in in vivo settings, and provided valuable and novel information on its molecular machinery. In conclusion, we make the case for intravital microscopy being a mature technique that can be used to investigate the molecular machinery of several intracellular events under physiological conditions

Intravital Two-Photon Microscopy for Studying the Uptake and Trafficking of Fluorescently Conjugated Molecules in Live Rodents

Here we describe an experimental system based on intravital two-photon microscopy for studying endocytosis in live animals. The rodent submandibular glands were chosen as model organs since they can be exposed easily, imaged without compromising their function and, furthermore, they are amenable to pharmacological and genetic manipulations. We show that the fibroblasts within the stroma of the glands readily internalize systemically injected molecules such as fluorescently conjugated dextran and bovine serum albumin, providing a robust model to study endocytosis. We dynamically image the trafficking of these probes from the early endosomes to the late endosomes and lysosomes while also visualizing homotypic fusion events between early endosomes. Finally, we demonstrate that pharmacological agents can be delivered specifically to the submandibular salivary glands thus providing a powerful tool to study the molecular machinery regulating endocytosis in a physiological context

A RhoA-FRET Biosensor Mouse for Intravital Imaging in Normal Tissue Homeostasis and Disease Contexts.

The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level. In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton

In Vivo Tissue-wide Synchronization of Mitochondrial Metabolic Oscillations

Little is known about the spatiotemporal coordination of mitochondrial metabolism in multicellular organisms in situ. Using intravital microscopy in live animals, we report that mitochondrial metabolism undergoes rapid and periodic oscillations under basal conditions. Notably, mitochondria in vivo behave as a network of functionally coupled oscillators, which maintain a high level of coordination throughout the tissue via the activity of gap junctions. These findings reveal a unique aspect of the relationship between tissue architecture and self-organization of mitochondrial metabolism in vivo