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We have identified novel actin filaments defined by tropomyosin Tpm4.2 at the ER. EM analysis of mouse embryo fibroblasts (MEFs) isolated from mice expressing a mutant Tpm4.2 (Tpm4(Plt53/Plt53)), incapable of incorporating into actin filaments, revealed swollen ER structures compared with wild-type (WT) MEFs (Tpm4(+/+)). ER-to-Golgi, but not Golgi-to-ER trafficking was altered in the Tpm4(Plt53/Plt53) MEFs following the transfection of the temperature sensitive ER-associated ts045-VSVg construct. Exogenous Tpm4.2 was able to rescue the ER-to-Golgi trafficking defect in the Tpm4(Plt53/Plt53) cells. The treatment of WT MEFs with the myosin II inhibitor, blebbistatin, blocked the Tpm4.2-dependent ER-to-Golgi trafficking. The lack of an effect on ER-to-Golgi trafficking following treatment of MEFs with CK666 indicates that branched Arp2/3-containing actin filaments are not involved in anterograde vesicle trafficking. We propose that unbranched, Tpm4.2-containing filaments have an important role in maintaining ER/Golgi structure and that these structures, in conjunction with myosin II motors, mediate ER-to-Golgi trafficking.

Background Geleophysic dysplasia (GPHYSD) is a disorder characterized by dysmorphic features, stiff joints and cardiac involvement due to defects of TGF‐β signaling. GPHYSD can be caused by mutations in FBN1, ADAMTLS2, and LTBP3 genes. Methods and Results Consistent with previous reports, we found intracellular inclusions of unknown material by electron microscopy (EM) in skin fibroblasts of two GPHYSD individuals carrying FBN1 mutations. Moreover, we found that the storage material is enclosed within lysosomes and is associated with the upregulation of several lysosomal genes. Treatment of GPHYSD fibroblasts carrying FBN1 mutations with the angiotensin II receptor type 1 inhibitor losartan that inhibits TGF‐β signaling did not reduce the storage but improved the extracellular deposition of fibrillin‐1 microfibrils. Conclusion Losartan is a promising candidate drug for treatment of GPHYSD due to FBN1 defects.

Sphingomyelin and cholesterol can assemble into domains and segregate from other lipids in the membranes. These domains are reported to function as platforms for protein transport and signalling. Do similar domains exist in the Golgi membranes and are they required for protein secretion? We tested this hypothesis by using D-ceramide-C6 to manipulate lipid homeostasis of the Golgi membranes. Lipidomics of the Golgi membranes isolated from D-ceramide-C6-treated HeLa cells revealed an increase in the levels of C6-sphingomyelin, C6-glucosylceramide, and diacylglycerol. D-ceramide-C6 treatment in HeLa cells inhibited transport carrier formation at the Golgi membranes without affecting the fusion of incoming carriers. The defect in protein secretion as a result of D-ceramide-C6 treatment was alleviated by knockdown of the sphingomyelin synthases 1 and 2. C6-sphingomyelin prevented liquid-ordered domain formation in giant unilamellar vesicles and reduced the lipid order in the Golgi membranes of HeLa cells. These findings highlight the importance of a regulated production and organization of sphingomyelin in the biogenesis of transport carriers at the Golgi membranes.

We have isolated a membrane fraction enriched in a class of transport carriers that form at the trans Golgi network (TGN) and are destined for the cell surface in HeLa cells. Protein kinase D (PKD) is required for the biogenesis of these carriers that contain myosin II, Rab6a, Rab8a, and synaptotagmin II, as well as a number of secretory and plasma membrane-specific cargoes. Our findings reveal a requirement for myosin II in the migration of these transport carriers but not in their biogenesis per se. Based on the cargo secreted by these carriers we have named them CARTS for CARriers of the TGN to the cell Surface. Surprisingly, CARTS are distinct from the carriers that transport vesicular stomatitis virus (VSV)-G protein and collagen I from the TGN to the cell surface. Altogether, the identification of CARTS provides a valuable means to understand TGN to cell surface traffic.

Mutations in the phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) 5-phosphatase OCRL cause Lowe syndrome, which is characterised by congenital cataracts, central hypotonia, and renal proximal tubular dysfunction. Previous studies have shown that OCRL interacts with components of the endosomal machinery; however, its role in endocytosis, and thus the pathogenic mechanisms of Lowe syndrome, have remained elusive. Here, we show that via its 5-phosphatase activity, OCRL controls early endosome (EE) function. OCRL depletion impairs the recycling of multiple classes of receptors, including megalin (which mediates protein reabsorption in the kidney) that are retained in engorged EEs. These trafficking defects are caused by ectopic accumulation of PtdIns4,5P2 in EEs, which in turn induces an N-WASP-dependent increase in endosomal F-actin. Our data provide a molecular explanation for renal proximal tubular dysfunction in Lowe syndrome and highlight that tight control of PtdIns4,5P2 and F-actin at the EEs is essential for exporting cargoes that transit this compartment.

Partial Retraction of: The EMBO Journal (2010) 29: 3607-3620. DOI: 10.1038/emboj.2010.237 | Published online 24 September 2010 Journal statement The journal contacted the authors in February 2022 about potential image insertions and duplications in Fig 4A and 4E. In the absence of source data, the authors are retracting Fig 4A, the lower panel of Fig 4E (LAMP1 immunoblot), and the following statements in the text that rely on these data: "Quantitative analysis showed that the percentage of Flotillin-1 associated with DRMs was increased in LSD endolysosomal membranes (Figure 4A), indicating an increased amount of cholesterol-enriched regions in these membrane samples." "LAMP1 also displayed a similar distribution profile in WT and LSD cells (Figure 4E)". Author statement The authors could not verify the aberrations in panel A of Fig 4 and the lower immunoblot (LAMP1) of 4E because the original source data are no longer available (12 years after publication, which is beyond the institute's 10-year data retention policy). The authors wish to clarify that the main conclusions of the paper are not affected by the retraction of Figure panels 4A and 4E for the following reasons: Figure panel 4A supports the observation that there are increased cholesterol-enhanced regions in LSD samples. This finding is also supported by data provided in figs 4B, 4C and 4D. Figure panel 4E: The LAMP1 blot in Fig 4E shows that the distribution of protein normally excluded from DRMs is not altered between Wt and LSD samples. This result is also supported by the upper blot in this panel (Transferrin receptor). The authors apologize for these errors and agree with this corrigendum; no response could be obtained from AL.

The particular compositions of the intracellular membrane organelles rely on the proteins and lipids received frequently through membrane trafficking. The delivery of these molecules is driven by the membrane-bound organelles known as transport carriers (TCs). Advanced microscopy approaches have revealed that TC morphology ranges from small vesicles to complex tubular membrane structures. These tubular TCs (TTCs) support effectively both sorting and transport events within the biosynthetic and endocytic pathways, while a coherent picture of the processes that define the formation and further fate of TTCs is still missing. Here, we present an overview of the mechanisms operating during the TTC life cycle, as well as of the emerging role of tubular carriers in different intracellular transport routes.

The delivery of mannose 6-phosphate receptors carrying lysosomal hydrolases from the trans-Golgi network (TGN) to the endosomal system is mediated by selective incorporation of the receptor-hydrolase complexes into vesicular transport carriers (TCs) that are coated with clathrin and the adaptor proteins, GGA and AP-1. Previous electron microscopy (EM) and biochemical studies have shown that these TCs consist of spherical coated vesicles with a diameter of 60-100 nm. The use of fluorescent live cell imaging, however, has revealed that at least some of this transport relies on a subset of apparently larger and highly pleiomorphic carriers that detach from the TGN and translocate toward the peripheral cytoplasm until they meet with distally located endosomes. The ultrastructure of such long-range TCs has remained obscure because of the inability to examine by conventional EM the morphological details of rapidly moving organelles. The recent development of correlative light-EM has now allowed us to obtain ultrastructural 'snapshots' of these TCs immediately after their formation from the TGN in live cells. This approach has revealed that such carriers range from typical 60- to 100-nm clathrin-coated vesicles to larger, convoluted tubular-vesicular structures displaying several coated buds. We propose that this subset of TCs serve as vehicles for long-range distribution of biosynthetic or recycling cargo from the TGN to the peripheral endosomes.

The Eps15 homology (EH) domain-containing protein, EHD1, has recently been ascribed a role in the recycling of receptors internalized by clathrin-mediated endocytosis. A subset of plasma membrane proteins can undergo internalization by a clathrin-independent pathway regulated by the small GTP-binding protein ADP-ribosylation factor 6 (Arf6). Here, we report that endogenous EHD proteins, as well as transgenic tagged EHD1, are associated with long, membrane-bound tubules containing Arf6. EHD1 appears to induce tubule formation, which requires nucleotide cycling on Arf6 and intact microtubules. Mutations in the N-terminal P-loop domain or deletion of the C-terminal EH domain of EHD1 prevent association of EHD1 with tubules or induction of tubule formation. The EHD1 tubules contain internalized major histocompatibility complex class I (MHC-I) molecules that normally traffic through the Arf6 pathway. Recycling assays show that overexpression of EHD1 enhances MHC-I recycling. These observations suggest an additional function of EHD1 as a tubule-inducing factor in the Arf6 pathway for recycling of plasma membrane proteins internalized by clathrin-independent endocytosis.