Regulators of the secretory pathway have distinct inputs into single-celled branching morphogenesis and seamless tube formation in the Drosophila trachea

Biological tubes serve as conduits through which gas, nutrients and other important fluids are delivered to tissues. Most biological tubes consist of multiple cells connected by epithelial junctions. Unlike these multicellular tubes, seamless tubes are unicellular and lack junctions. Seamless tubes are present in various organ systems, including the vertebrate vasculature, C.elegans excretory system, and Drosophila tracheal system. The Drosophila tracheal system is a network of air-filled tubes that delivers oxygen to all tissues. Specialized cells within the tracheal system, called terminal cells, branch extensively and form seamless tubes. Terminal tracheal tubes are polarized; the lumenal membrane has apical identity whereas the outer membrane exhibits basal characteristics. Although various aspects of membrane trafficking have been implicated in terminal cell morphogenesis, the precise secretory pathway requirements for basal and apical membrane growth have yet to be elucidated. In the present study, we demonstrate that anterograde trafficking, retrograde trafficking and Golgi-to-plasma membrane vesicle fusion are each required for the complex branched architecture of the terminal cell, but their inputs during seamless lumen formation are more varied. The COPII subunit, Sec 31, and ER exit site protein, Sec16, are critical for subcellular tube architecture, whereas the SNARE proteins Syntaxin 5, Syntaxin 1 and Syntaxin15 are required for seamless tube growth and maintenance. These data suggest that distinct components of the secretory pathway have differential contributions to basal and apical membrane growth and maintenance during terminal cell morphogenesis.

Inclusion membrane growth and composition is altered by overexpression of specific Incs in Chlamydia trachomatis L2

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections. This obligate intracellular bacterium develops within a membrane-bound vacuole called an inclusion, which sequesters the chlamydiae from the host cytoplasm. Host-pathogen interactions at this interface are mediated by chlamydial inclusion membrane proteins (Incs). However, the specific functions of most Incs are poorly characterized. Previous work from our labs indicated that expressing an IncF fusion protein at high levels in C. trachomatis L2 negatively impacted inclusion expansion and progeny production. We hypothesize that some Incs function in the structure and organization of the inclusion membrane and that overexpression of those Incs will alter the composition of endogenous Incs within the inclusion membrane. Consequently, inclusion biogenesis and chlamydial development is negatively impacted. To investigate this, C. trachomatis L2 was transformed with inducible expression plasmids encoding incF-, ct813-, or ct226-FLAG. Overexpression of IncF-FLAG or CT813-FLAG, but not CT226-FLAG, altered chlamydial development as demonstrated by smaller inclusions, fewer progeny, and increased plasmid loss. The overexpression of CT813-FLAG reduced the detectable levels of endogenous IncE and IncG in the inclusion membrane. Notably, recruitment of sorting nexin-6, a eukaryotic protein binding partner of IncE, was also reduced after CT813 overexpression. Gene expression studies and ultrastructural analysis of chlamydial organisms demonstrated that chlamydial development was altered when CT813-FLAG was overexpressed. Overall, these data indicate that disrupting the expression of specific Incs changed the composition of Incs within the inclusion membrane and the recruitment of associated host cell proteins, which negatively impacted C. trachomatis development.

Hydroxylated sphingolipid biosynthesis regulates photoreceptor apical domain morphogenesis

Apical domains of epithelial cells often undergo dramatic changes during morphogenesis to form specialized structures, such as microvilli. Here, we addressed the role of lipids during morphogenesis of the rhabdomere, the microvilli-based photosensitive organelle of Drosophila photoreceptor cells. Shotgun lipidomics analysis performed on mutant alleles of the polarity regulator crumbs, exhibiting varying rhabdomeric growth defects, revealed a correlation between increased abundance of hydroxylated sphingolipids and abnormal rhabdomeric growth. This could be attributed to an up-regulation of fatty acid hydroxylase transcription. Indeed, direct genetic perturbation of the hydroxylated sphingolipid metabolism modulated rhabdomere growth in a crumbs mutant background. One of the pathways targeted by sphingolipid metabolism turned out to be the secretory route of newly synthesized Rhodopsin, a major rhabdomeric protein. In particular, altered biosynthesis of hydroxylated sphingolipids impaired apical trafficking via Rab11, and thus apical membrane growth. The intersection of lipid metabolic pathways with apical domain growth provides a new facet to our understanding of apical growth during morphogenesis.

Pex24 and Pex32 are required to tether peroxisomes to the ER for organelle biogenesis, positioning and segregation in yeast

ABSTRACTThe yeast Hansenula polymorpha contains four members of the Pex23 family of peroxins, which characteristically contain a DysF domain. Here we show that all four H. polymorpha Pex23 family proteins localize to the endoplasmic reticulum (ER). Pex24 and Pex32, but not Pex23 and Pex29, predominantly accumulate at peroxisome–ER contacts. Upon deletion of PEX24 or PEX32 – and to a much lesser extent, of PEX23 or PEX29 – peroxisome–ER contacts are lost, concomitant with defects in peroxisomal matrix protein import, membrane growth, and organelle proliferation, positioning and segregation. These defects are suppressed by the introduction of an artificial peroxisome–ER tether, indicating that Pex24 and Pex32 contribute to tethering of peroxisomes to the ER. Accumulation of Pex32 at these contact sites is lost in cells lacking the peroxisomal membrane protein Pex11, in conjunction with disruption of the contacts. This indicates that Pex11 contributes to Pex32-dependent peroxisome–ER contact formation. The absence of Pex32 has no major effect on pre-peroxisomal vesicles that occur in pex3 atg1 deletion cells.

Autophagosome biogenesis: From membrane growth to closure

Autophagosome biogenesis involves de novo formation of a membrane that elongates to sequester cytoplasmic cargo and closes to form a double-membrane vesicle (an autophagosome). This process has remained enigmatic since its initial discovery >50 yr ago, but our understanding of the mechanisms involved in autophagosome biogenesis has increased substantially during the last 20 yr. Several key questions do remain open, however, including, What determines the site of autophagosome nucleation? What is the origin and lipid composition of the autophagosome membrane? How is cargo sequestration regulated under nonselective and selective types of autophagy? This review provides key insight into the core molecular mechanisms underlying autophagosome biogenesis, with a specific emphasis on membrane modeling events, and highlights recent conceptual advances in the field.

Transcytosis via the late endocytic pathway as a cell morphogenetic mechanism

Plasma membranes fulfil many physiological functions. In polarized cells, different membrane compartments take on specialized roles, each being allocated correct amounts of membrane. The Drosophila tracheal system, an established tubulogenesis model, contains branched terminal cells with subcellular tubes formed by apical plasma membrane invagination. We show that apical endocytosis and late endosome-mediated trafficking determine the membrane allocation to the apical and basal membrane domains. Basal plasma membrane growth stops if endocytosis is blocked, whereas the apical membrane grows excessively. Plasma membrane is initially delivered apically, and then continuously endocytosed, together with apical and basal cargo. We describe an organelle carrying markers of late endosomes and multivesicular bodies (MVBs) that is abolished by inhibiting endocytosis, and which we suggest acts as transit station for membrane destined to be redistributed both apically and basally. This is based on the observation that disrupting MVB formation prevents growth of both compartments.

A perinuclear microtubule-organizing center controls nuclear positioning and basement membrane secretion

Non-centrosomal microtubule-organizing centers (ncMTOCs) have a variety of roles presumed to serve the diverse functions of the range of cell types in which they are found. ncMTOCs are diverse in their composition, subcellular localization, and function. Here we report a novel perinuclear MTOC in Drosophila fat body cells that is anchored by Msp300/Nesprin at the cytoplasmic surface of the nucleus. Msp300 recruits the MT minus-end protein Patronin/CAMSAP, which functions redundantly with Ninein to further recruit the MT polymerase Msps/XMAP215 to assemble non-centrosomal MTs and does so independently of the widespread MT nucleation factor γ-tubulin. Functionally, the fat body ncMTOC and the radial MT arrays it organizes is essential for nuclear positioning and for secretion of basement membrane components via retrograde dynein-dependent endosomal trafficking that restricts plasma membrane growth. Together, this study identifies a perinuclear ncMTOC with unique architecture and MT regulation properties that serves vital functions.HighlightsA novel perinuclear MTOC in differentiated fat body cellsThe predominant nucleator, γ-tubulin, is not required at the fat body ncMTOCMsp300/Nesprin organizes the ncMTOC at the nuclear surface by recruiting Patronin/CAMSAP and the spectraplakin ShotPatronin cooperates with Ninein to control MT assembly at the fat body ncMTOC by recruiting MspsMsps, a MT polymerase, is essential for radial MT elongation from the fat body ncMTOCPatronin and Msps associateThe ncMTOC and radial MTs, but not actin, control nuclear positioning in the fat bodyThe fat body MTOC controls retrograde endocytic trafficking to regulate plasma membrane growth and secretion of basement membrane proteins

Conserved Ark1-related kinases control TORC2 signaling

In all orders of life, cell cycle progression is dependent upon cell growth, and the extent of growth required for cell cycle progression is proportional to growth rate. Thus, cells growing rapidly in rich nutrients are substantially larger than slow growing cells. In budding yeast, a conserved signaling network surrounding Tor complex 2 (TORC2) controls growth rate and cell size in response to nutrient availability. Here, a search for new components of the TORC2 network identified a pair of redundant kinase paralogs called Ark1 and Prk1. Previous studies found that Ark/Prk play roles in endocytosis. Here, we show that Ark/Prk are embedded in the TORC2 network, where they appear to influence TORC2 signaling independently of their roles in endocytosis. We also show that reduced endocytosis leads to increased cell size, which indicates that cell size homeostasis requires coordinated control of plasma membrane growth and endocytosis. The discovery that Ark/Prk are embedded in the TORC2 network suggests a model in which TORC2-dependent signals control both plasma membrane growth and endocytosis, which would ensure that the rates of each process are matched to each other and to the availability of nutrients so that cells achieve and maintain an appropriate size.