Probing the Role of the Actin Cytoskeleton During Regulated Exocytosis by Intravital Microscopy

Abstract

Summary The actin cytoskeleton plays a fundamental role in controlling several steps during regulated exocytosis. Here we describe a combination of procedures that are aimed at studying the dynamics and the mechanism of the actin cytoskeleton in the salivary glands of live rodents, a model for exocrine secretion. Our approach relies on intravital microscopy, an imaging technique that enables imaging biological events in live animals at a subcellular resolution, and it is complemented by the use of pharmacological agents and indirect immunofluorescence in the salivary tissue.

Imaging membrane remodeling during regulated exocytosis in live mice

“…For example, individual exocytic events have been visualized in explanted and cultured lacrimal glands [19], in Xenopus eggs during fertilization [26–28], in isolated pancreatic acini [20, 21, 34–36], in primary-cultured type II pneumocytes cells [22–24], and in the Weibel-Palade bodies in endothelial cells [25]. A combination of pharmacological and genetic approaches has revealed ground-breaking information about the basic machinery regulating secretion, ranging from the role of specific components of the actin cytoskeleton, to the characterization of the signaling pathways, to the role of specific lipids [17, 19, 22–25, 27, 28, 34, 37]. …”

“…In order to image the dynamics of secretory granules during regulated exocytosis, our group has taken advantage of a transgenic mouse strain that expresses GFP in the cytosol [3, 33, 37]. Since the fluorescent molecule is not sorted into the secretory pathway, the secretory granules appear as discrete vesicles, devoid of GFP, and clustered in close proximity to the APM [3] (Fig.…”

“…1B, arrows). This model is very robust and can be used to characterize the mechanisms of degranulation in vivo, and to accurately measure the kinetics of individual exocytic events [37] (Fig. 1A and 1B).…”

“…We took two approaches. The first is based on the transient non viral-mediated transfection of plasmids encoding for Lifeact tagged with either GFP or RFP [3, 4, 33, 37, 42]. The second is based on the use of transgenic mice expressing these probes [33, 37] (Fig.…”

Cdc42 negatively regulates endocytosis during apical plasma membrane maintenance and development in mouse tubular organs in vivo

“…Here, we used a combination of intravital subcellular microscopy (ISMic), a light microscopy-based technique that enables imaging the dynamics of intracellular structures in live animals (Masedunskas et al, 2011a;Masedunskas et al, 2013;Milberg et al, 2014;Pittet and Weissleder, 2011;Weigert et al, 2013), and indirect immunofluorescence, to examine the role of Cdc42 in controlling maintenance and formation of the intercellular canaliculi (IC) in the acinar cells of the submandibular salivary glands (SMGs) in live mice. These structures are narrow tubes, formed by the APM of two adjacent acinar cells (Fig.1), which constitute a network spanning throughout the secretory acini ( Fig.…”