Lisa M. Hartnell

Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor

The cation-independent mannose 6-phosphate receptor (CI-MPR) mediates sorting of lysosomal hydrolase precursors from the TGN to endosomes. After releasing the hydrolase precursors into the endosomal lumen, the unoccupied receptor returns to the TGN for further rounds of sorting. Here, we show that the mammalian retromer complex participates in this retrieval pathway. The hVps35 subunit of retromer interacts with the cytosolic domain of the CI-MPR. This interaction probably occurs in an endosomal compartment, where most of the retromer is localized. In particular, retromer is associated with tubular–vesicular profiles that emanate from early endosomes or from intermediates in the maturation from early to late endosomes. Depletion of retromer by RNA interference increases the lysosomal turnover of the CI-MPR, decreases cellular levels of lysosomal hydrolases, and causes swelling of lysosomes. These observations indicate that retromer prevents the delivery of the CI-MPR to lysosomes, probably by sequestration into endosome-derived tubules from where the receptor returns to the TGN.

Ggas: A Family of Adp Ribosylation Factor-Binding Proteins Related to Adaptors and Associated with the Golgi Complex

Formation of intracellular transport intermediates and selection of cargo molecules are mediated by protein coats associated with the cytosolic face of membranes. Here, we describe a novel family of ubiquitous coat proteins termed GGAs, which includes three members in humans and two in yeast. GGAs have a modular structure consisting of a VHS domain, a region of homology termed GAT, a linker segment, and a region with homology to the ear domain of γ-adaptins. Immunofluorescence microscopy showed colocalization of GGAs with Golgi markers, whereas immunoelectron microscopy of GGA3 revealed its presence on coated vesicles and buds in the area of the TGN. Treatment with brefeldin A or overexpression of dominant-negative ADP ribosylation factor 1 (ARF1) caused dissociation of GGAs from membranes. The GAT region of GGA3 was found to: target a reporter protein to the Golgi complex; induce dissociation from membranes of ARF-regulated coats such as AP-1, AP-3, AP-4, and COPI upon overexpression; and interact with activated ARF1. Disruption of both GGA genes in yeast resulted in impaired trafficking of carboxypeptidase Y to the vacuole. These observations suggest that GGAs are components of ARF-regulated coats that mediate protein trafficking at the TGN.

A tubular EHD1‐containing compartment involved in the recycling of major histocompatibility complex class I molecules to the plasma membrane

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.

The GGAs Promote ARF-Dependent Recruitment of Clathrin to the TGN

The GGAs constitute a family of modular adaptor-related proteins that bind ADP-ribosylation factors (ARFs) and localize to the trans-Golgi network (TGN) via their GAT domains. Here, we show that binding of the GAT domain stabilizes membrane-bound ARF1.GTP due to interference with the action of GTPase-activating proteins. We also show that the hinge and ear domains of the GGAs interact with clathrin in vitro, and that the GGAs promote recruitment of clathrin to liposomes in vitro and to TGN membranes in vivo. These observations suggest that the GGAs could function to link clathrin to membrane-bound ARF.GTP.

Direct visualization of Escherichia coli chemotaxis receptor arrays using cryo-electron microscopy

Signal transduction in bacterial chemotaxis is initiated by the binding of extracellular ligands to a specialized family of methyl-accepting chemoreceptor proteins. Chemoreceptors cluster at distinct regions of the cell and form stable ternary complexes with the histidine autokinase CheA and the adapter protein CheW. Here we report the direct visualization and spatial organization of chemoreceptor arrays in intact Escherichia coli cells by using cryo-electron tomography and biochemical techniques. In wild-type cells, ternary complexes are arranged as an extended lattice, which may or may not be ordered, with significant variations in the size and specific location among cells in the same population. In the absence of CheA and CheW, chemoreceptors do not form observable clusters and are diffusely localized to the cell pole. At disproportionately high receptor levels, membrane invaginations containing nonfunctional, axially interacting receptor assemblies are formed. However, functional chemoreceptor arrays can be reestablished by increasing cellular levels of CheA and CheW. Our results demonstrate that chemotaxis in E. coli requires the presence of chemoreceptor arrays and that the formation of these arrays requires the scaffolding interactions of the signaling molecules CheA and CheW.

3D visualization of HIV transfer at the virological synapse between dendritic cells and T cells

The efficiency of HIV infection is greatly enhanced when the virus is delivered at conjugates between CD4+ T cells and virus-bearing antigen-presenting cells such as macrophages or dendritic cells via specialized structures known as virological synapses. Using ion abrasion SEM, electron tomography, and superresolution light microscopy, we have analyzed the spatial architecture of cell-cell contacts and distribution of HIV virions at virological synapses formed between mature dendritic cells and T cells. We demonstrate the striking envelopment of T cells by sheet-like membrane extensions derived from mature dendritic cells, resulting in a shielded region for formation of virological synapses. Within the synapse, filopodial extensions emanating from CD4+ T cells make contact with HIV virions sequestered deep within a 3D network of surface-accessible compartments in the dendritic cell. Viruses are detected at the membrane surfaces of both dendritic cells and T cells, but virions are not released passively at the synapse; instead, virus transfer requires the engagement of T-cell CD4 receptors. The relative seclusion of T cells from the extracellular milieu, the burial of the site of HIV transfer, and the receptor-dependent initiation of virion transfer by T cells highlight unique aspects of cell-cell HIV transmission.

Human Vam6p promotes lysosome clustering and fusion in vivo

Regulated fusion of mammalian lysosomes is critical to their ability to acquire both internalized and biosynthetic materials. Here, we report the identification of a novel human protein, hVam6p, that promotes lysosome clustering and fusion in vivo. Although hVam6p exhibits homology to the Saccharomyces cerevisiae vacuolar protein sorting gene product Vam6p/Vps39p, the presence of a citron homology (CNH) domain at the NH2 terminus is unique to the human protein. Overexpression of hVam6p results in massive clustering and fusion of lysosomes and late endosomes into large (2–3 μm) juxtanuclear structures. This effect is reminiscent of that caused by expression of a constitutively activated Rab7. However, hVam6p exerts its effect even in the presence of a dominant-negative Rab7, suggesting that it functions either downstream of, or in parallel to, Rab7. Data from gradient fractionation, two-hybrid, and coimmunoprecipitation analyses suggest that hVam6p is a homooligomer, and that its self-assembly is mediated by a clathrin heavy chain repeat domain in the middle of the protein. Both the CNH and clathrin heavy chain repeat domains are required for induction of lysosome clustering and fusion. This study implicates hVam6p as a mammalian tethering/docking factor characterized with intrinsic ability to promote lysosome fusion in vivo.

Ion-Abrasion Scanning Electron Microscopy Reveals Surface-Connected Tubular Conduits in HIV-Infected Macrophages

HIV-1-containing internal compartments are readily detected in images of thin sections from infected cells using conventional transmission electron microscopy, but the origin, connectivity, and 3D distribution of these compartments has remained controversial. Here, we report the 3D distribution of viruses in HIV-1-infected primary human macrophages using cryo-electron tomography and ion-abrasion scanning electron microscopy (IA-SEM), a recently developed approach for nanoscale 3D imaging of whole cells. Using IA-SEM, we show the presence of an extensive network of HIV-1-containing tubular compartments in infected macrophages, with diameters of ∼150–200 nm, and lengths of up to ∼5 µm that extend to the cell surface from vesicular compartments that contain assembling HIV-1 virions. These types of surface-connected tubular compartments are not observed in T cells infected with the 29/31 KE Gag-matrix mutant where the virus is targeted to multi-vesicular bodies and released into the extracellular medium. IA-SEM imaging also allows visualization of large sheet-like structures that extend outward from the surfaces of macrophages, which may bend and fold back to allow continual creation of viral compartments and virion-lined channels. This potential mechanism for efficient virus trafficking between the cell surface and interior may represent a subversion of pre-existing vesicular machinery for antigen capture, processing, sequestration, and presentation.

Nonsense Mutations in ADTB3A Cause Complete Deficiency of the β3A Subunit of Adaptor Complex-3 and Severe Hermansky-Pudlak Syndrome Type 2

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disease consisting of oculocutaneous albinism and a storage pool deficiency resulting from absent platelet dense bodies. The disorder is genetically heterogeneous. The majority of patients, including members of a large genetic isolate in northwest Puerto Rico, have mutations in HPS1. Another gene, ADTB3A, was shown to cause HPS-2 in two brothers having compound heterozygous mutations that allowed for residual production of the gene product, the β3A subunit of adaptor complex-3 (AP-3). This heterotetrameric complex serves as a coat protein–mediating formation of intracellular vesicles, e.g. the melanosome and platelet dense body, from membranes of the trans-Golgi network. We determined the genomic organization of the human ADTB3A gene, with intron/exon boundaries, and describe a third patient with β3A deficiency. This 5-y-old boy has two nonsense mutations, C1578T (R→X) and G2028T (E→X), which produce no ADTB3A mRNA and no β3A protein. The associated μ3 subunit of AP-3 is also entirely absent. In fibroblasts, the cell biologic concomitant of this deficiency is robust and aberrant trafficking through the plasma membrane of LAMP-3, an integral lysosomal membrane protein normally carried directly to the lysosome. The clinical concomitant is a severe, G-CSF–responsive neutropenia in addition to oculocutaneous albinism and platelet storage pool deficiency. Our findings expand the molecular, cellular, and clinical spectrum of HPS-2 and call for an increased index of suspicion for this diagnosis among patients with features of albinism, bleeding, and neutropenia.

A new variant of Hermansky-Pudlak syndrome due to mutations in a gene responsible for vesicle formation

Hermansky-Pudlak syndrome is a recessive type of oculocutaneous albinism that is prevalent in northwest Puerto Rico due to a founder effect (1–3). In this syndrome, bleeding and bruising occur because of the absence of platelet dense bodies, which normally release serotonin, calcium, and adenosine diphosphate to trigger a secondary aggregation response (4). In addition, the accumulation of a lipid-protein complex called ceroid lipofuscin (5,6) is thought to cause the pulmonary fibrosis (7) and granulomatous colitis (8) seen in this disease. One gene causing Hermansky-Pudlak syndrome, HPS-1, encodes a 700 amino acid protein of unknown function (9 –11). Northwest Puerto Rican patients are homozygous for a 16 base pair (bp) duplication in HPS-1, but most non-Puerto Rican patients have no mutations in this gene (12,13). Consequently, it has become accepted that several different genes, when mutated, can cause Hermansky-Pudlak syndrome (12–14). This phenomenon, called locus heterogeneity, is also found in mice: 14 different mouse strains manifest a type of Hermansky-Pudlak syndrome (pigment dilution and platelet storage pool deficiency), each due to a different gene (15). To date, three of these genes have been cloned. Pale ear is the murine analogue of patients with HPS-1 mutations (16,17), and pearl (18) and mocha (19) have defects in adaptor complex-3 (AP-3). One protein subunit of adaptor complex-3, called b3A, is mutated in the pearl mouse, while another protein subunit, called d, is mutated in the mocha mouse. Adaptor complex-3 is an aggregate of four different peptides and serves as a “coat” protein that concentrates in a donor membrane and recruits other membrane components to become part of a newly formed vesicle. These vesicles, such as lysosomes and peroxisomes, are functional compartments that provide an optimal environment for specialized biochemical processes. Adaptor complex-3 is thought to be responsible for the formation of pigment-forming vesicles (melanosomes) and platelet storage vesicles (dense bodies) (20,21). We describe two brothers with Hermansky-Pudlak syndrome with mutations in the b3A subunit of adaptor complex-3 (22,23).