Phagolysosome resolution requires contacts with the endoplasmic reticulum and phosphatidylinositol-4-phosphate signalling.

Phosphoinositides have a pivotal role in the maturation of nascent phagosomes into microbicidal phagolysosomes. Following degradation of their contents, mature phagolysosomes undergo resolution, a process that remains largely uninvestigated. Here we studied the role of phosphoinositides in phagolysosome resolution. Phosphatidylinositol-4-phosphate (PtdIns(4)P), which is abundant in maturing phagolysosomes, was depleted as they tubulated and resorbed. Depletion was caused, in part, by transfer of phagolysosomal PtdIns(4)P to the endoplasmic reticulum, a process mediated by oxysterol-binding protein-related protein 1L (ORP1L), a RAB7 effector. ORP1L formed discrete tethers between the phagolysosome and the endoplasmic reticulum, resulting in distinct regions with alternating PtdIns(4)P depletion and enrichment. Tubules emerged from PtdIns(4)P-rich regions, where ADP-ribosylation factor-like protein 8B (ARL8B) and SifA- and kinesin-interacting protein/pleckstrin homology domain-containing family M member 2 (SKIP/PLEKHM2) accumulated. SKIP binds preferentially to monophosphorylated phosphoinositides, of which PtdIns(4)P is most abundant in phagolysosomes, contributing to their tubulation. Accordingly, premature hydrolysis of PtdIns(4)P impaired SKIP recruitment and phagosome resolution. Thus, resolution involves phosphoinositides and tethering of phagolysosomes to the endoplasmic reticulum.

Cytoskeleton: septins do the horizontal tango.

Septins are a family of membrane-associated filamentous proteins that are essential in many biological processes, such as cell division. A recent study has provided the first visualization of septin filament formation in real time, leading to important new insights into their organization.

Characterization of presynaptic septin complexes in mammalian hippocampal neurons.

Septins are GTPases that form heteromeric complexes and are linked to neurological disorders. Although several septin subcomplexes have been reported in various mammalian tissues, the cellular and subcellular distribution of these complexes is largely unexplored. Using antibodies against ten mammalian septins, we show that septins diverge with respect to their tissue distribution implying that septin complexes in various tissues have unique composition. Although all ten septins examined were expressed in brain tissue, we describe septin complex(es) including SEPT3, SEPT5, SEPT6, SEPT7 and SEPT11 that could be functional within the presynapse because, unlike other septins they: (1) showed an increase in expression from embryonic day 15 to post-natal day 70, (2) were abundantly expressed in axons and growth cones of developing hippocampal neurons, (3) were found in presynaptic terminals of mature synapses, (4) were enriched in a preparation of synaptic vesicles and (5) immunoprecipitated together from brain tissue and cultured nerve cells. Knockdown of SEPT5 or SEPT7 in developing hippocampal neurons impaired axon growth. Because septins are functionally linked to the cytoskeleton and vesicle traffic, presynaptic neuronal septin complexes could be important for ensuring proper axon development and neurotransmitter release.

Mammalian septins are required for phagosome formation.

Septins are members of a highly conserved family of filamentous proteins that are required in many organisms for the completion of cytokinesis. In addition, septins have been implicated in a number of important cellular processes and have been suggested to have roles in regulating membrane traffic. Given the proposed role of septins in cell membrane dynamics, we investigated the function of septins during FcgammaR-mediated phagocytosis. We show that several septins are expressed in RAW264.7 and J774 mouse macrophage cell lines and that SEPT2 and SEPT11 are colocalized with submembranous actin-rich structures during the early stages of FcgammaR-mediated phagocytosis. In addition, SEPT2 accumulation is seen in primary human neutrophils and in nonprofessional phagocytes. The time course of septin accumulation mirrors actin accumulation and is inhibited by latrunculin and genistein, but not other inhibitors of phagocytosis. Inhibition of septin function by transient expression of the BD3 domain of BORG3, known to cause septin aggregation, or depletion of SEPT2 or SEPT11 by RNAi, significantly inhibited FcgammaR-mediated phagocytosis of IgG-coated latex beads. Interestingly, this occurred without affecting the accumulation of actin or the actin-associated protein coronin-1. These observations show that, although not necessary for actin recruitment, septins are required for efficient FcgammaR-mediated phagocytosis.