JCB_201612126 1925..1936

Membrane remodeling is essential for the regulation of a variety of events, ranging from organelle biogenesis to cell migration, cell division, and membrane trafficking. This process requires both continuous modifications of the composition and the properties of the lipid bilayer (Shnyrova et al., 2009; Kozlov et al., 2014) and the application of mechanical forces directly to the membranes. The forces are provided by cytoplasmic protein complexes (Daley and Yamada, 2013; Henne et al., 2013; Simunovic and Bassereau, 2014), which include curvature generators such as BAR (Bin/Amphiphysin/Rvs) domain–containing proteins (Kozlov et al., 2014; Suetsugu et al., 2014), the ESC RT (endosomal sorting complexes required for transport) complex (Henne et al., 2013; Chiaruttini et al., 2015), fission factors such as the large GTPase dynamin (Ferguson and De Camilli, 2012), and various components of the actomyosin cytoskeleton (Hatch et al., 2014; Korobova et al., 2014). Although membrane remodeling has been extensively studied in cell-free systems and cultured cells, information about the dynamics, mechanisms, and molecular players that drive and regulate this process in multicellular organisms in vivo, where the three-dimensional architecture, cell–cell interactions, and cues coming from vasculature and nervous systems likely have key effects (Shitara and Weigert, 2015), remains sparse. To address this, we established an in vivo model system to study the role of the actomyosin cytoskeleton in membrane remodeling during regulated exocytosis, a fundamental process in secretory organs (Burgoyne and Morgan, 2003; Sokac et al., 2003; Jerdeva et al., 2005; Nightingale et al., 2011; Miklavc et al., 2012; Porat-Shliom et al., 2013). Using intravital subcellular microscopy (ISMic), a light microscopy–based technique that enables imaging intracellular structures in live animals (Pittet and Weissleder, 2011; Weigert et al., 2013), we previously investigated the remodeling of secretory granules in the acinar cells of rodent submandibular salivary glands (SGs; Masedunskas et al., 2011a; Milberg et al., 2014). We showed that, after the granules release their contents into the extracellular space, their membranes gradually integrate into the apical plasma membrane (APM). This remodeling process is triggered by the assembly of an actomyosin complex comprising F-actin and two isoforms of the actin-binding motor nonmuscle myosin II, NMI IA and NMI IB (Fig. 1 A; Masedunskas et al., 2011a). Using a pharmacological approach, we determined that both F-actin assembly and NMII contractile activity were required to drive the integraMembrane remodeling plays a fundamental role during a variety of biological events. However, the dynamics and the molecular mechanisms regulating this process within cells in mammalian tissues in situ remain largely unknown. In this study, we use intravital subcellular microscopy in live mice to study the role of the actomyosin cytoskeleton in driving the remodeling of membranes of large secretory granules, which are integrated into the plasma membrane during regulated exocytosis. We show that two isoforms of nonmuscle myosin II, NMI IA and NMI IB, control distinct steps of the integration process. Furthermore, we find that F-actin is not essential for the recruitment of NMII to the secretory granules but plays a key role in the assembly and activation of NMII into contractile filaments. Our data support a dual role for the actomyosin cytoskeleton in providing the mechanical forces required to remodel the lipid bilayer and serving as a scaffold to recruit key regulatory molecules. Concerted actions of distinct nonmuscle myosin II isoforms drive intracellular membrane remodeling in live animals