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A polarized arrangement of neuronal microtubule arrays is the foundation of membrane trafficking and subcellular compartmentalization. Conserved among both invertebrates and vertebrates, axons contain exclusively “plus-end-out” microtubules while dendrites contain a high percentage of “minus-end-out” microtubules, the origins of which have been a mystery. Here we show that the dendritic growth cone contains a non-centrosomal microtubule organizing center (ncMTOC), which generates minus-end-out microtubules along outgrowing dendrites and plus-end-out microtubules in the growth cone. RAB-11-positive recycling endosomes accumulate in this region and are responsible for localizing the microtubule nucleation complex γ-TuRC. The MTOC tracks the extending growth cone by kinesin-1/UNC-116-mediated endosome movements on distal plus-end-out microtubules and dynein-mediated endosome clustering near MTOC. Critically, perturbation of the function or localization of the MTOC causes reversed microtubule polarity in dendrites. These findings unveil the dendritic MTOC as a critical organelle for establishing axon-dendrite polarity.

Summary Gene inactivation by transposon insertion or allelic exchange is a powerful approach to probe gene function. Unfortunately, many microbes, including Chlamydia , are not amenable to routine molecular genetic manipulations. Here we describe an arrayed library of chemically induced mutants of the genetically intransigent pathogen Chlamydia trachomatis , in which all mutations have been identified by whole-genome sequencing, providing a platform for reverse genetic applications. An analysis of possible loss-of-function mutations in the collection uncovered plasticity in the central metabolic properties of this obligate intracellular pathogen. We also describe the use of the library in a forward genetic screen that identified InaC as a bacterial factor that binds host ARF and 14-3-3 proteins and modulates F-actin assembly and Golgi redistribution around the pathogenic vacuole. This work provides a robust platform for reverse and forward genetic approaches in Chlamydia and should serve as a valuable resource to the community.

The secreted Chlamydia protease CPAF cleaves a defined set of mammalian and Chlamydia proteins in vitro. As a result, this protease has been proposed to modulate a range of bacterial and host cellular functions. However, it has recently come into question the extent to which many of its identified substrates constitute bona fide targets of proteolysis in infected host cell rather than artifacts of postlysis degradation. Here, we clarify the role played by CPAF in cellular models of infection by analyzing Chlamydia trachomatis mutants deficient for CPAF activity. Using reverse genetic approaches, we identified two C. trachomatis strains possessing nonsense, loss of function mutations in cpa (CT858) and a third strain containing a mutation in type II secretion (T2S) machinery that inhibited CPAF activity by blocking zymogen secretion and subsequent proteolytic maturation into the active hydrolase. HeLa cells infected with T2S− or CPAF−C. trachomatis mutants lacked detectable in vitro CPAF proteolytic activity and were not defective for cellular traits that have been previously attributed to CPAF activity, including resistance to staurosporine induced apoptosis, Golgi fragmentation, altered NFκB dependent gene expression, and resistance to reinfection. However, CPAF deficient mutants did display impaired generation of infectious elementary bodies (EBs), indicating an important role for this protease in the full replicative potential of C. trachomatis. In addition, we provide compelling evidence in live cells that CPAF mediated protein processing of at least two host protein targets, vimentin filaments and the nuclear envelope protein lamin associated protein 1 (LAP1), occurs rapidly after the loss of the inclusion membrane integrity, but before loss of plasma membrane permeability and cell lysis. CPAF dependent processing of host proteins correlates with a loss of inclusion membrane integrity, and so we propose that CPAF plays a role late in infection, possibly during the stages leading to the dismantling of the infected cell prior to the release of EBs during cell lysis. Fil: Snavely, Emily. University of Duke; Estados Unidos Fil: Kokes, Marcela. University of Duke; Estados Unidos Fil: Dunn, Joe Dan. University of Duke; Estados Unidos Fil: Saka, Hector Alex. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of Duke; Estados Unidos Fil: Nguyen, Bidong D.. University of Duke; Estados Unidos Fil: Bastidas, Robert J.. University of Duke; Estados Unidos Fil: McCafferty, Dewey G.. University of Duke; Estados Unidos Fil: Valdivia, Raphael H.. University of Duke; Estados Unidos

All aspects of Chlamydia survival are intimately linked to the cell biology of its host. Recent advances in cell biological techniques and new tools to perform loss-of-function experiments in mammalian cells have accelerated one's understanding of the extent to which Chlamydia manipulates the host. Homotypic fusion of C. trachomatis inclusions could serve to consolidate resources and reduce competition among multiple growing inclusions. It is clear that Incs on early inclusions likely play important roles in remodeling the nascent inclusion to segregate from the endolysosomal pathway and maintain single inclusion morphology in fusogenic Chlamydia species. However, the role played by soluble effectors secreted early or even during entry should not be discounted when considering early interactions with host cell biology. Of host sphingolipids (SLs), only sphingomyelin, and not glucosylceramide, is delivered to the chlamydial inclusion , suggesting highly specific interactions with host pathways. Multivesicular bodies (MVBs) are late endocytic compartments in which the limiting membrane of endosomes has invaginated into the lumen to form intraluminal vesicles containing membrane proteins destined for degradation. Much of the focus of investigations into chlamydial anti-immune strategies has centered on the interruption of innate immune signaling pathways. Given the long evolutionary history of the association of Chlamydia spp. with eukaryotic cells, these bacteria are expected to reveal new insights into basic aspects of eukaryotic cell biology, primordial mechanisms of cell autonomous innate immunity, and novel pathogenic strategies.