Recent advances in imaging single protein mobility

Recent advances in imaging single protein mobility and organization in live organisms such as Drosophila larva motor nerve terminals have initiated this by providing a useful platform to investigate how the dynamic changes in the actomyosin network correlates with vesicle docking and fusion in vivo22, 91. Use of dual-camera imaging to combine synaptic vesicle tracking [92] with changes in Xylometazoline HCl dynamics will also elucidate the relevance of changes in the tensile strength of the cortical actin network in staging vesicle docking and fusion [93]. It is worth noting that the cortical actin network is known to be actively remodeled in response to elevated calcium ion concentration. It is interesting to note that catecholamines as well as other types of secretion are only mediated by Ca2+ (see Outstanding Questions) and that other secretagogues and downstream signaling platforms can lead to exocytosis such as cAMP, ATP, pituitary adenylate cyclase-activating polypeptide (PACAP), and chloroquine 94, 95, 96, 97. Secretagogue stimulation by either barium chloride or nicotine have been shown to reorganize the actomyosin network 12, 98. It would be interesting in the future to understand how the cortical actin network is coupled to these other forms of stimulation. Furthermore, a number of signaling pathways prevents nicotine induced catecholamine secretion through an unknown mechanism [99]. It would be interesting to check whether the cortical actin network could be altered by these antagonistic signaling pathways.
Introduction Endothelial cells acutely respond to inflammation and vascular injury by the signal-dependent release of immune-modulatory and hemostatic molecules to the cell surface and the external environment. Therefore, they are equipped with specific secretory organelles, most importantly the endothelial-specific Weibel-Palade bodies (WPBs). WPBs are elongated cigar-shaped organelles, 0.5–5 μm in length and 0.1–0.3 μm in diameter, that originate at the trans-Golgi network (TGN) and share properties with lysosome-related organelles. One of the main constituent of these granules is the large multimeric glycoprotein von-Willebrand factor (VWF) that is organized together with its propeptide (VWF-proregion) into characteristic compact tubules longitudinally arranged in the WPBs [[1], [2], [3], [4], [5]]. WPBs also store an important pro-inflammatory transmembrane protein, the leukocyte adhesion receptor P-selectin, which is sorted to WPBs by the binding of its luminal domain to VWF [6,7]. Activation of endothelial cells induces the fusion of WPBs with the plasma membrane and their content release into the blood and sub-endothelium [2,8]. However, it appears that WPBs can respond differently to different stimuli and this can lead to a selective release of WPB cargo. Most likely, WPBs can secrete their contents in various ways: they can fully collapse into the plasma membrane as single organelles and release all of their cargo [9,10], and they can undergo long lingering kiss [11], multigranular/compound [12,13], and/or even cumulative exocytosis [14]. Moreover, it has been reported that after fusion, WPBs linger at the plasma membrane as empty vacuoles from where membrane is retrieved by compensatory endocytosis [14]. However, the specific conditions and the kinetics of these processes remain elusive, in particular when different stimuli are considered, as VWF release is a very fast process after treatment with the secretagogues histamine or forskolin, but rather slow following PMA stimulation [[9], [10], [11],[14], [15], [16]]. The actin cortex and cortical actin rearrangements could function in different ways during WPB exocytosis. The actin cortex could constitute a significant barrier to exocytosing WPBs but actin could also promote WPB fusion and cargo release, as discussed controversially in the recent past [15,[17], [18], [19]]. Before fusion, WPBs are anchored at the cortical cytoskeleton via a Rab27a/MyRIP/Myosin Va complex, preventing premature WPB exocytosis and allowing for final VWF and thus WPB maturation [20,21]. How exactly this cortical anchorage and therefore an inhibitory role of the actin cortex on WPB exocytosis is regulated remains elusive, although the relative WPB occupancy of positive versus negative Rab27a effectors, Slp4a versus MyRIP, appears to play an important role [22]. After WPB fusion with the plasma membrane, it has been suggested that the large sticky WPB cargo, VWF, needs an additional force to be expelled outwards and that this force is provided by a highly dynamic actomyosin ring or coat which is recruited to the bottom of fused WPBs. This was shown to be important following PMA and probably also forskolin and to some extent histamine stimulation [15,19,23,24]. However, another report using histamine or ionomycin stimulation to induce Ca2+-dependent WPB exocytosis did not observe the formation of actin rings/coats at fused WPB and could not confirm an involvement of the actomyosin machinery in VWF release [17]. Thus, the role of the actin cytoskeleton at late steps of WPB exocytosis, i.e. cargo release following the actual plasma membrane fusion, remains not fully resolved.