SignificanceThe molecular mechanism for sealing newly formed nuclear envelopes was unclear until the recent discovery that endosomal sorting complexes required for transport III (ESCRT-III) proteins mediate this process. Cmp7p (CHMP7), in particular, was identified as an early acting factor that recruits other ESCRT-III proteins to the nuclear envelope. A fundamental aspect of the varied roles of ESCRT factors is their recruitment by site-specific adaptors, yet the central question of how the ESCRT machinery is targeted to nuclear membranes has remained outstanding. Our study identifies the inner nuclear membrane protein LEM2 as a key, conserved factor that recruits CHMP7 and downstream ESCRT-III proteins to breaches in the nuclear envelope.

ESCRT-III proteins have been implicated in sealing the nuclear envelope in mammals, spindle pole body dynamics in fission yeast, and surveillance of defective nuclear pore complexes in budding yeast. Here, we report that Lem2p (LEM2), a member of the LEM (Lap2-Emerin-Man1) family of inner nuclear membrane proteins, and the ESCRT-II/ESCRT-III hybrid protein Cmp7p (CHMP7), work together to recruit additional ESCRT-III proteins to holes in the nuclear membrane. In S. pombe, deletion of the ATPase vps4 leads to severe defects in nuclear morphology and integrity. These phenotypes are suppressed by loss-of-function mutations that arise spontaneously in lem2 or cmp7, implying that these proteins may function upstream in the same pathway. Building on these genetic interactions, we explored the role of LEM2 during nuclear envelope reformation in human cells. We found that CHMP7 and LEM2 enrich at the same region of the chromatin disc periphery during this window of cell division, and that CHMP7 can bind directly to the C-terminal domain of LEM2 in vitro. We further found that, during nuclear envelope formation, recruitment of the ESCRT factors CHMP7, CHMP2A and IST1/CHMP8 all depend on LEM2 in human cells. We conclude that Lem2p/LEM2 is a conserved nuclear sitespecific adaptor that recruits Cmp7p/CHMP7 and downstream ESCRT factors to the nuclear envelope.. CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint .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/10.1101/049312 doi: bioRxiv preprint first posted online Apr. 19, 2016; 3 SignificanceThe molecular mechanism for sealing newly formed nuclear envelopes was unclear until the recent discovery that ESCRT-III proteins mediate this process. CHMP7, in particular, was identified as an early-acting factor that recruits other ESCRT-III proteins to the nuclear envelope.A fundamental aspect of the varied roles of ESCRT factors is their recruitment by site-specific adaptors, yet the central question of how the ESCRT machinery is targeted to nuclear membranes has remained outstanding. Our study identifies the inner nuclear membrane protein LEM2 as a key, conserved factor that recruits CHMP7 and downstream ESCRT-III proteins to breaches in the nuclear envelope.

Branching morphogenesis, the process by which cells or tissues generate tree-like networks that function to increases surface area or in contacting multiple targets, is a common developmental motif in multicellular organisms. We use Drosophila tracheal terminal cells, a component of the insect respiratory system, to investigate branching morphogenesis that occurs on the single cell level. Here, we show that the exocyst, a conserved protein complex that facilitates docking and tethering of vesicles at the plasma membrane, is required for terminal cell branch outgrowth. We find that exocyst-deficient terminal cells have highly truncated branches and show an accumulation of vesicles within their cytoplasm and are also defective in subcellular lumen formation. We also show that vesicle trafficking pathways mediated by the Rab GTPases Rab10 and Rab11 are redundantly required for branch outgrowth. In terminal cells, the PAR-polarity complex is required for branching, and we find the PAR complex is required for proper membrane localization of the exocyst, thus identifying a molecular link between the branching and outgrowth programs. Together, our results suggest a model where exocyst mediated vesicle trafficking facilitates branch outgrowth, while de novo branching requires cooperation between the PAR and exocyst complexes.

Cellular tubes have diverse morphologies, including multicellular, unicellular and subcellular architectures. Subcellular tubes are found prominently within the vertebrate vasculature, the insect breathing system and the nematode excretory apparatus, but how such tubes form is poorly understood. To characterize the cellular mechanisms of subcellular tube formation, we have refined methods of high pressure freezing/freeze substitution to prepare Drosophila larvae for transmission electron microscopic (TEM) analysis. Using our methods, we have found that subcellular tube formation may proceed through a previously undescribed multimembrane intermediate composed of vesicles bound within a novel subcellular compartment. We have also developed correlative light/TEM procedures to identify labeled cells in TEM-fixed larval samples. Using this technique, we have found that Vacuolar ATPase (V-ATPase) and the V-ATPase regulator Rabconnectin-3 are required for subcellular tube formation, probably in a step resolving the intermediate compartment into a mature lumen. In general, our ultrastructural analysis methods could be useful for a wide range of cellular investigations in Drosophila larvae.

Transient receptor potential canonical 1 (TRPC1) protein is abundantly expressed in cardiomyocytes. While TRPC1 is supposed to be critically involved in cardiac hypertrophy, its physiological role in cardiomyocytes is poorly understood. We investigated the subcellular location of TRPC1 and its contribution to Ca 2+ signaling in mammalian ventricular myocytes. Immunolabeling, three-dimensional scanning confocal microscopy and quantitative colocalization analysis revealed an abundant intracellular location of TRPC1 in neonatal rat ventricular myocytes (NRVMs) and adult rabbit ventricular myocytes. TRPC1 was colocalized with intracellular proteins including sarco/endoplasmic reticulum Ca 2+ ATPase 2 in the sarcoplasmic reticulum (SR). Colocalization with wheat germ agglutinin, which labels the glycocalyx and thus marks the sarcolemma including the transverse tubular system, was low. Super-resolution and immunoelectron microscopy supported the intracellular location of TRPC1. We investigated Ca 2+ signaling in NRVMs after adenoviral TRPC1 overexpression or silencing. In NRVMs bathed in Na + and Ca 2+ free solution, TRPC1 overexpression and silencing was associated with a decreased and increased SR Ca 2+ content, respectively. In isolated rabbit cardiomyocytes bathed in Na + and Ca 2+ free solution, we found an increased decay of the cytosolic Ca 2+ concentration [Ca 2+ ] i and increased SR Ca 2+ content in the presence of the TRPC channel blocker SKF-96365. In a computational model of rabbit ventricular myocytes at physiological pacing rates, Ca 2+ leak through SR TRPC channels increased the systolic and diastolic [Ca 2+ ] i with only minor effects on the action potential and SR Ca 2+ content. Our studies suggest that TRPC1 channels are localized in the SR, and not present in the sarcolemma of ventricular myocytes. The studies provide evidence for a role of TRPC1 as a contributor to SR Ca 2+ leak in cardiomyocytes, which was previously explained by ryanodine receptors only. We propose that the findings will guide us to an understanding of TRPC1 channels as modulators of [Ca 2+ ] i and contractility in cardiomyocytes.