Charles Ferguson

Endosome-to-cytosol transport of viral nucleocapsids

During viral infection, fusion of the viral envelope with endosomal membranes and nucleocapsid release were thought to be concomitant events. We show here that for the vesicular stomatitis virus they occur sequentially, at two successive steps of the endocytic pathway. Fusion already occurs in transport intermediates between early and late endosomes, presumably releasing the nucleocapsid within the lumen of intra-endosomal vesicles, where it remains hidden. Transport to late endosomes is then required for the nucleocapsid to be delivered to the cytoplasm. This last step, which initiates infection, depends on the late endosomal lipid lysobisphosphatidic acid (LBPA) and its putative effector Alix/AIP1, and is regulated by phosphatidylinositol-3-phosphate (PtdIns(3)P) signalling via the PtdIns(3)P-binding protein Snx16. We conclude that the nucleocapsid is exported into the cytoplasm after the back-fusion of internal vesicles with the limiting membrane of late endosomes, and that this process is controlled by the phospholipids LBPA and PtdIns(3)P and their effectors.

Caveolin-1 is essential for liver regeneration

Liver regeneration is an orchestrated cellular response that coordinates cell activation, lipid metabolism, and cell division. We found that caveolin-1 gene–disrupted mice (cav1–/– mice) exhibited impaired liver regeneration and low survival after a partial hepatectomy. Hepatocytes showed dramatically reduced lipid droplet accumulation and did not advance through the cell division cycle. Treatment of cav1–/– mice with glucose (which is a predominant energy substrate when compared to lipids) drastically increased survival and reestablished progression of the cell cycle. Thus, caveolin-1 plays a crucial role in the mechanisms that coordinate lipid metabolism with the proliferative response occurring in the liver after cellular injury.

Dynamic microtubules regulate the local concentration of E-cadherin at cell-cell contacts.

In contrast to the well-established relationship between cadherins and the actin cytoskeleton, the potential link between cadherins and microtubules (MTs) has been less extensively investigated. We now identify a pool of MTs that extend radially into cell-cell contacts and are inhibited by manoeuvres that block the dynamic activity of MT plus-ends (e.g. in the presence of low concentrations of nocodazole and following expression of a CLIP-170 mutant). Blocking dynamic MTs perturbed the ability of cells to concentrate and accumulate E-cadherin at cell-cell contacts, as assessed both by quantitative immunofluorescence microscopy and fluorescence recovery after photobleaching (FRAP) analysis, but did not affect either transport of E-cadherin to the plasma membrane or the amount of E-cadherin expressed at the cell surface. This indicated that dynamic MTs allow cells to concentrate E-cadherin at cell-cell contacts by regulating the regional distribution of E-cadherin once it reaches the cell surface. Importantly, dynamic MTs were necessary for myosin II to accumulate and be activated at cadherin adhesive contacts, a mechanism that supports the focal accumulation of E-cadherin. We propose that this population of MTs represents a novel form of cadherin-MT cooperation, where cadherin adhesions recruit dynamic MTs that, in turn, support the local concentration of cadherin molecules by regulating myosin II activity at cell-cell contacts.

Rab23, a negative regulator of hedgehog signaling, localizes to the plasma membrane and the endocytic pathway.

The regulation of hedgehog signaling by vesicular trafficking was exemplified by the finding that Rab23, a Rab‐GTPase vesicular transport protein, is mutated in open brain mice. In this study, the localization of Rab23 was analyzed by light and immunoelectron microscopy after expression of wild‐type (Rab23‐GFP), constitutively active Rab23 (Rab23Q68L‐GFP), and inactive Rab23 (Rab23S23N‐GFP) in a range of mammalian cell types. Rab23‐GFP and Rab23Q68L‐GFP were predominantly localized to the plasma membrane but were also associated with intracellular vesicular structures, whereas Rab23S23N‐GFP was predominantly cytosolic. Vesicular Rab23‐GFP colocalized with Rab5Q79L and internalized transferrin‐biotin, but not with a marker of the late endosome or the Golgi complex. To investigate Rab23 with respect to members of the hedgehog signaling pathway, Rab23‐GFP was coexpressed with either patched or smoothened. Patched colocalized with intracellular Rab23‐GFP but smoothened did not. Analysis of patched distribution by light and immunoelectron microscopy revealed it is primarily localized to endosomal elements, including transferrin receptor‐positive early endosomes and putative endosome carrier vesicles and, to a lesser extent, with LBPA‐positive late endosomes, but was excluded from the plasma membrane. Neither patched or smoothened distribution was altered in the presence of wild‐type nor mutant Rab23‐GFP, suggesting that despite the endosomal colocalization of Rab23 and patched, it is likely that Rab23 acts more distally in regulating hedgehog signaling.

Regulation of Albumin Endocytosis by PSD95/Dlg/ZO-1 (PDZ) Scaffolds INTERACTION OF Na+-H+ EXCHANGE REGULATORY FACTOR-2 WITH ClC-5

The constitutive reuptake of albumin from the glomerular filtrate by receptor-mediated endocytosis is a key function of the renal proximal tubules. Both the Cl– channel ClC-5 and the Na+-H+ exchanger isoform 3 are critical components of the macromolecular endocytic complex that is required for albumin uptake, and therefore the cell-surface levels of these proteins may limit albumin endocytosis. This study was undertaken to investigate the potential roles of the epithelial PDZ scaffolds, Na+-H+ exchange regulatory factors, NHERF1 and NHERF2, in albumin uptake by opossum kidney (OK) cells. We found that ClC-5 co-immunoprecipitates with NHERF2 but not NHERF1 from OK cell lysate. Experiments using fusion proteins demonstrated that this was a direct interaction between an internal binding site in the C terminus of ClC-5 and the PDZ2 module of NHERF2. In OK cells, NHERF2 is restricted to the intravillar region while NHERF1 is located in the microvilli. Silencing NHERF2 reduced both cell-surface levels of ClC-5 and albumin uptake. Conversely, silencing NHERF1 increased cell-surface levels of ClC-5 and albumin uptake, presumably by increasing the mobility of NHE3 in the membrane and its availability to the albumin uptake complex. Surface biotinylation experiments revealed that both NHERF1 and NHERF2 were associated with the plasma membrane and that NHERF2 was recruited to the membrane in the presence of albumin. The importance of the interaction between NHERF2 and the cytoskeleton was demonstrated by a significant reduction in albumin uptake in cells overexpressing an ezrin binding-deficient mutant of NHERF2. Thus NHERF1 and NHERF2 differentially regulate albumin uptake by mechanisms that ultimately alter the cell-surface levels of ClC-5.

Patched1 is required in neural crest cells for the prevention of orofacial clefts

Defects such as cleft lip with or without cleft palate (CL/P) are among the most common craniofacial birth defects in humans. In many cases, the underlying molecular and cellular mechanisms that result in these debilitating anomalies remain largely unknown. Perturbed hedgehog (HH) signalling plays a major role in craniofacial development, and mutations in a number of pathway constituents underlie craniofacial disease. In particular, mutations in the gene encoding the major HH receptor and negative regulator, patched1 (PTCH1), are associated with both sporadic and familial forms of clefting, yet relatively little is known about how PTCH1 functions during craniofacial morphogenesis. To address this, we analysed the consequences of conditional loss of Ptch1 in mouse neural crest cell-derived facial mesenchyme. Using scanning electron microscopy (SEM) and live imaging of explanted facial primordia, we captured defective nasal pit invagination and CL in mouse embryos conditionally lacking Ptch1. Our analysis demonstrates interactions between HH and FGF signalling in the development of the upper lip, and reveals cell-autonomous and non-autonomous roles mediated by Ptch1. In particular, we show that deletion of Ptch1 in the facial mesenchyme alters cell morphology, specifically in the invaginating nasal pit epithelium. These findings highlight a critical link between the neural crest cells and olfactory epithelium in directing the morphogenesis of the mammalian lip and nose primordia. Importantly, these interactions are critically dependent on Ptch1 function for the prevention of orofacial clefts.

LINKS IN THE CHAIN: THE CONTRIBUTION OF KETTIN TO THE ELASTICITY OF INSECT MUSCLES

Asynchronous flight muscle fibers are activated by periodic stretches and need to be stiff for strain to be transmitted to the contractile system. Kettin associated with thin filaments and projectin with thick filaments contribute to fiber stiffness. Kettin extends along thin filaments with the N-terminus in the Z-disc and the C-terminus outside. C filaments connecting thick filaments to the Z-disc contain projectin but not kettin. Insect flight myofibrils have a titin PEVK epitope which is only exposed on stretch, suggesting it is short and inaccessible. It is concluded that kettin stiffens thin filaments near the Z-disc and projectin and titin provide elasticity to C filaments.

Domains of yeast plasma membrane and ATPase-associated glycoprotein

In yeast homogenates the plasma membrane H(+)-ATPase and a major surface glycoprotein of about 115 kDa are present in two membrane fractions with peak densities in sucrose gradients of 1.17 and 1.22. Immunogold electron microscopy of frozen yeast sections indicates that the ATPase is exclusively (greater than 95%) present at the surface membrane. Therefore the two ATPase-containing fractions appear to correspond to different domains of the plasma membrane. The 115 kDa glycoprotein is tightly associated with the ATPase during solubilization and purification of the enzyme. However, in a mutant lacking the glycoprotein the activity of the plasma membrane H(+)-ATPase is similar to wild type, suggesting that this association is fortuitous. The ATPase and the glycoprotein are difficult to separate by electrophoresis and therefore binding of concanavalin A to the ATPase cannot be unambiguously demonstrated in wild-type yeast. By utilizing the mutant without glycoprotein it was shown that the ATPase band of 105 kDa binds concanavalin A.

ORP5 and ORP8 bind phosphatidylinositol-4, 5-biphosphate (PtdIns(4,5)P 2) and regulate its level at the plasma membrane.

ORP5 and ORP8, members of the oxysterol-binding protein (OSBP)-related proteins (ORP) family, are endoplasmic reticulum membrane proteins implicated in lipid trafficking. ORP5 and ORP8 are reported to localize to endoplasmic reticulum–plasma membrane junctions via binding to phosphatidylinositol-4-phosphate (PtdIns(4)P), and act as a PtdIns(4)P/phosphatidylserine counter exchanger between the endoplasmic reticulum and plasma membrane. Here we provide evidence that the pleckstrin homology domain of ORP5/8 via PtdIns(4,5)P2, and not PtdIns(4)P binding mediates the recruitment of ORP5/8 to endoplasmic reticulum–plasma membrane contact sites. The OSBP-related domain of ORP8 can extract and transport multiple phosphoinositides in vitro, and knocking down both ORP5 and ORP8 in cells increases the plasma membrane level of PtdIns(4,5)P2 with little effect on PtdIns(4)P. Overall, our data show, for the first time, that phosphoinositides other than PtdIns(4)P can also serve as co-exchangers for the transport of cargo lipids by ORPs.ORP5/8 are endoplasmic reticulum (ER) membrane proteins implicated in lipid trafficking that localize to ER-plasma membrane (PM) contacts and maintain membrane homeostasis. Here the authors show that PtdIns(4,5)P2 plays a critical role in the targeting and function of ORP5/8 at the PM.

Caveolin 1 restricts Group A Streptococcus invasion of nonphagocytic host cells

Caveolae are composed of 2 major proteins, caveolin 1 (CAV1) and cavin 1 or polymerase transcript release factor I (CAVIN1). Here, we demonstrate that CAV1 levels modulate invasion of Group A Streptococcus (GAS) into nonphagocytic mammalian cells. GAS showed enhanced internalisation into CAV1‐knockout mouse embryonic fibroblasts and CAV1 knockdown human epithelial HEp‐2 cells, whereas overexpression of CAV1 in HEp‐2 cells reduced GAS invasion. This effect was not dependent on the expression of the GAS fibronectin binding protein SfbI, which had previously been implicated in caveolae‐mediated uptake. Nor was this effect dependent on CAVIN1, as knockout of CAVIN1 in mouse embryonic fibroblasts resulted in reduced GAS internalisation. Although CAV1 restricted GAS invasion into host cells, we observed only minimal association of invading GAS (strain M1T15448) with CAV1 by immunofluorescence and very low association of invading M1T15448 with caveolae by transmission electron microscopy. These observations suggest that physical interaction with caveolae is not needed for CAV1 restriction of invading GAS. An indirect mechanism of action is also consistent with the finding that changing membrane fluidity reverses the increased invasion observed in CAV1‐null cells. Together, these results suggest that CAV1 protects host cells against GAS invasion by a caveola‐independent mechanism.