The exocyst is a conserved octameric complex that tethers exocytic vesicles to the plasma membrane prior to fusion. Exocyst assembly and delivery mechanisms remain unclear, especially in mammalian cells. Here we tagged multiple endogenous exocyst subunits with sfGFP or Halo using Cas9 gene-editing, to create single and double knock-in lines of mammary epithelial cells, and interrogated exocyst dynamics by high-speed imaging and correlation spectroscopy. We discovered that mammalian exocyst is comprised of tetrameric subcomplexes that can associate independently with vesicles and plasma membrane and are in dynamic equilibrium with octamer and monomers. Membrane arrival times are similar for subunits and vesicles, but with a small delay (~80msec) between subcomplexes. Departure of SEC3 occurs prior to fusion, whereas other subunits depart just after fusion. About 9 exocyst complexes are associated per vesicle. These data reveal the mammalian exocyst as a remarkably dynamic two-part complex and provide important insights into assembly/disassembly mechanisms.

The exocyst is a conserved octameric complex that tethers exocytic vesicles to the plasma membrane prior to fusion. Exocyst assembly and delivery mechanisms remain unclear, especially in mammalian cells. Here we tagged multiple endogenous exocyst subunits with sfGFP or Halo using Cas9 gene editing, to create single and double knock-in lines of mammary epithelial cells, and interrogated exocyst dynamics by high-speed imaging and correlation spectroscopy. We discovered that mammalian exocyst is comprised of tetrameric subcomplexes that, unexpectedly, can associate independently with vesicles and plasma membrane and are in dynamic equilibrium. Membrane arrival times are similar for subunits and vesicles, but with a small delay (~80msec) between subcomplexes. Departure of Sec3 occurs prior to fusion, whereas other subunits depart just after fusion. Single molecule counting indicates ~9 exocyst complexes associated per vesicle. These data reveal the mammalian exocyst as a remarkably dynamic two-part complex and provide important new insights into assembly/disassembly mechanisms.Key words: vesicles, membrane fusion, dynamics, TIRFM, CRISPR, gene-editing, protein complex.. CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/354449 doi: bioRxiv preprint first posted online Jun. 22, 2018; 3 Traffic between membrane-bound compartments requires the docking of cargo vesicles at target membranes, and their subsequent fusion through the interactions of SNARE proteins. The capture and fusion of vesicles are both promoted by molecular tethers known as multisubunit tethering complexes 1 . One group of such tethers, sometimes called CATCHR (complexes associate with tethering containing helical rods) comprises multisubunit complexes required for fusion in the secretory pathway, and includes COG, Dsl1p, GARP and the exocyst 2 . The endolysosomal pathway contains two different tethering complexes, CORVET and HOPS, with similar overall structures to the CATCHR group 3 .COG consists of two subcomplexes, each containing four subunits, which function together within the Golgi 4-6 . The exocyst is also octameric, and is necessary for exocytic vesicle fusion to the plasma membrane (PM), but the organization of the complex has been controversial [7][8][9][10] . Several studies in yeast suggest that one (Sec3) or two (Sec3 and Exo70)subunits associate with the PM and recruit a vesicle-bound subcomplex of the other subunits, but other work argues that the exocyst consists of 2 subcomplexes of 4 subunits each that form a stable octamer or, in mammalian cells, that 5 subunits at the PM recruit 3 other subunits on the vesicle [11][12][13][14][15][16][17][18][19][20][21][22] . Rab GTPases promote exocyst binding to the vesicle, and SNARES, Rho family GTPases, the PAR3 polarity protein, and phospho-inositide binding domains are all involved ...

The epididymis is a male accessory organ and functions for sperm maturation and storage under the control of androgen. The development of the epididymis is also androgen dependent. The Wolffian duct (WD), anlagen of the epididymis, is formed in both male and female embryos; however, it is stabilized only in male embryos by testicular androgen. Androgen drives subsequent differentiation of the WD into the epididymis. Although the essential roles of androgen in WD masculinization and epididymal function have been established, little is known about cellular events regulated precisely by androgen signaling during these processes. It is also unclear whether androgen signaling, especially in the epithelia, has further function for epididymal epithelial cell differentiation. In this study we examined the cellular death and proliferation controlled by androgen signaling via the androgen receptor (AR) in WD stabilization. Analyses using AR knockout mice revealed that androgen signaling inhibits epithelial cell death in this process. Analysis of AP2α-Cre;AR(flox/Y) mice, in which AR function is deleted in the WD epithelium, revealed that epithelial AR is not required for the WD stabilization but is required for epithelial cell differentiation in the epididymis. Specifically, loss of epithelial AR significantly reduced expression of p63 that is essential for differentiation of basal cells in the epididymal epithelium. We also interrogated the possibility of regulation of the p63 gene (Trp63) by AR in vitro and found that p63 is a likely direct target of AR regulation.

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a type I transmembrane glycoprotein, is known as one of the most specific lymphatic vessel markers in the skin. In this study, we found that the ectodomain of LYVE-1 undergoes proteolytic cleavage, and this process produces soluble LYVE-1. We further identified the cleavage site for ectodomain shedding and generated an uncleavable mutant of LYVE-1. In lymphatic endothelial cells, ectodomain shedding of LYVE-1 was induced by vascular endothelial growth factor (VEGF)-A, an important factor for angiogenesis and lymphangiogenesis under pathological conditions. VEGF-A-induced LYVE-1 ectodomain shedding was mediated via the extracellular signal-regulated kinase (ERK) and a disintegrin and metalloproteinase (ADAM) 17. Wild-type LYVE-1, but not uncleavable LYVE-1, promoted migration of lymphatic endothelial cells in response to VEGF-A. Immunostaining analyses in human psoriasis skin lesions and VEGF-A transgenic mouse skin suggested that the ectodomain shedding of LYVE-1 occurred in lymphatic vessels undergoing chronic inflammation. These results indicate that the ectodomain shedding of LYVE-1 might be involved in promoting pathological lymphangiogenesis.Lymphatic vessels play crucial roles in maintaining tissue fluid homeostasis, immune surveillance, and fat absorption. Physiological lymphangiogenesis is regulated by several genes including Prox1 and vascular endothelial growth factor (VEGF)-C that induce the sprouting of lymphatic endothelial cells from embryonic veins during mammalian development (1, 2). Lymphatic endothelial cells express VEGF receptors (VEGFRs) 2 that represent a family of receptor tyrosine kinases. Among them, VEGFR-3 plays an essential role in promoting physiological lymphangiogenesis because VEGFR-3 shows a high affinity toward VEGF-C. In fact, several missense mutations are known among tyrosine kinase domains in Flt4/ VEGFR-3 that lead to the formation of Milroy disease, a congenital lymphedema due to an insufficient development of cutaneous lymphatic vessels. Thus, the VEGF-C/VEGFR-3 pathway plays a crucial role in promoting physiological and pathological lymphangiogenesis.VEGF-A, a specific ligand for VEGFR-1 and VEGFR-2, induces cutaneous angiogenesis in physiological and pathological conditions such as psoriasis (3). Targeted overexpression of mouse VEGF-A164 in the epidermis of transgenic mice leads to the formation of erythematous plaques resembling psoriasis (4), indicating that VEGF-A plays an important role in the pathogenesis of psoriasis. Our previous studies indicated that targeted overexpression of VEGF-A in mouse skin promotes the prominent enlargement of lymphatic vessels during acute inflammation by ultraviolet-B irradiation (5) and induces tumor-associated lymphangiogenesis as well as angiogenesis during multistep chemically induced skin carcinogenesis (6). Importantly, tumor lymphangiogenesis actively promotes enhanced metastasis to draining lymph nodes and beyond in VEGF-A transgenic mice as compared with wild-typ...