Lucy M. Collinson

Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy

Mammalian Atg9 (mAtg9) is a multispanning membrane protein that resides in a novel compartment. mAtg9 interacts dynamically with phagophores and forming autophagosomes. It is proposed that mAtg9 function is required to initiate autophagosome formation and increase the number of autophagosomes.

HIV-1 trafficking to the dendritic cell-T-cell infectious synapse uses a pathway of tetraspanin sorting to the immunological synapse

Dendritic cells (DCs) are essential components of the early events of HIV infection. Here, we characterized the trafficking pathways that HIV‐1 follows during its capture by DCs and its subsequent presentation to CD4+ T cells via an infectious synapse. Immunofluorescence microscopy indicates that the virus‐containing compartment in mature DCs (mDCs) co‐labels for the tetraspanins CD81, CD82, and CD9 but contains little CD63 or LAMP‐1. Using ratio imaging of pH‐reporting fluorescent virions in live DCs, we show that HIV‐1 is internalized in an intracellular endocytic compartment with a pH of 6.2. Significantly, we demonstrate that the infectivity of cell‐free virus is more stable at mildly acidic pH than at neutral pH. Using electron microscopy, we confirm that HIV‐1 accumulates in intracellular vacuoles that contain CD81 positive internal membranes but overlaps only partially with CD63. When allowed to contact T cells, HIV‐1‐loaded DCs redistribute CD81, and CD9, as well as internalized HIV‐1, but not the immunological synapse markers MHC‐II and T‐cell receptor to the infectious synapse. Together, our results indicate that HIV‐1 is internalized into a non‐conventional, non‐lysosomal, endocytic compartment in mDCs and further suggest that HIV‐1 is able to selectively subvert components of the intracellular trafficking machinery required for formation of the DC–T‐cell immunological synapse to facilitate its own cell‐to‐cell transfer and propagation.

Constitutive sharing of recycling synaptic vesicles between presynaptic boutons

The synaptic vesicle cycle is vital for sustained neurotransmitter release. It has been assumed that functional synaptic vesicles are replenished autonomously at individual presynaptic terminals. Here we tested this assumption by using FM dyes in combination with fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured rat hippocampal neurons. After photobleaching, synapses acquired recently recycled FM dye–labeled vesicles originating from nonphotobleached synapses by a process requiring dynamic actin turnover. The imported vesicles entered the functional pool at their host synapses, as revealed by the exocytic release of the dye upon stimulation. FM1-43 photoconversion and ultrastructural analysis confirmed the incorporation of imported vesicles into the presynaptic terminal, where they mixed with the native vesicle pools. Our results demonstrate that synaptic vesicle recycling is not confined to individual presynaptic terminals as is widely believed; rather, a substantial proportion of recycling vesicles are shared constitutively between boutons.

Functional redundancy of Rab27 proteins and the pathogenesis of Griscelli syndrome

Griscelli syndrome (GS) patients and the corresponding mouse model ashen exhibit defects mainly in two types of lysosome-related organelles, melanosomes in melanocytes and lytic granules in CTLs. This disease is caused by loss-of-function mutations in RAB27A, which encodes 1 of the 60 known Rab GTPases, critical regulators of vesicular transport. Here we present evidence that Rab27a function can be compensated by a closely related protein, Rab27b. Rab27b is expressed in platelets and other tissues but not in melanocytes or CTLs. Morphological and functional tests in platelets derived from ashen mice are all within normal limits. Both Rab27a and Rab27b are found associated with the limiting membrane of platelet-dense granules and to a lesser degree with alpha-granules. Ubiquitous transgenic expression of Rab27a or Rab27b rescues ashen coat color, and melanocytes derived from transgenic mice exhibit widespread peripheral distribution of melanosomes instead of the perinuclear clumping observed in ashen melanocytes. Finally, transient expression in ashen melanocytes of Rab27a or Rab27b, but not other Rabs, restores peripheral distribution of melanosomes. Our data suggest that Rab27b is functionally redundant with Rab27a and that the pathogenesis of GS is determined by the relative expression of Rab27a and Rab27b in specialized cell types.

The leaden Gene Product Is Required with Rab27a to Recruit Myosin Va to Melanosomes in Melanocytes

The function of lysosome‐related organelles such as melanosomes in melanocytes, and lytic granules in cytotoxic T lymphocytes is disrupted in Griscelli syndrome and related diseases. Griscelli syndrome results from loss of function mutations in either the RAB27A (type 1 Griscelli syndrome) or MYO5A (type 2 Griscelli syndrome) genes. Melanocytes from Griscelli syndrome patients and respective murine models ashen (Rab27a mutant), dilute (myosin Va mutant), and leaden exhibit perinuclear clustering of melanosomes. Recent work suggests that Rab27a is required to recruit myosin Va to melanosomes, thereby tethering melanosomes to the peripheral actin network and promoting melanosome retention at the tips of melanocytic dendrites. Here, we characterize the function of the leaden gene product. We show that Rab27a, but not myosin Va, can be localized to melanosomes in leaden melanocytes, suggesting that the leaden gene product acts downstream of, or in parallel to, Rab27a in melanocytes to promote recruitment of myosin Va to melanosomes. We also observed reduced levels of myosin Va protein in leaden and ashen melanocytes, suggesting that myosin Va stability is influenced by the leaden and ashen gene products. In leaden cytotoxic T lymphocytes, we observed that lytic granules polarize towards the immunological synapse and kill target cells normally. However, in contrast to melanocytes, we found that neither the leaden gene product (melanophilin) nor myosin Va was detectable in cytotoxic T lymphocytes. These results suggest that Rab27a interacts with different classes of effector proteins in melanocytes and cytotoxic T lymphocytes.

Exploring the third dimension: volume electron microscopy comes of age.

Groundbreaking advances in volume electron microscopy and specimen preparation are enabling the 3-dimensional visualisation of specimens with unprecedented detail, and driving a gratifying resurgence of interest in the ultrastructural examination of cellular systems. Serial section techniques, previously the domain of specialists, are becoming increasingly automated with the development of systems such as the automatic tape-collecting ultramicrotome, and serial blockface and focused ion beam scanning electron microscopes. These changes are rapidly broadening the scope of biomedical studies to which volume electron microscopy techniques can be applied beyond the brain. Further innovations in microscope design are also in the pipeline, which have the potential to enhance the speed and quality of data collection. The recent introduction of integrated light and electron microscopy systems will revolutionise correlative light and volume electron microscopy studies, by enabling the sequential collection of data from light and electron imaging modalities without intermediate specimen manipulation. In doing so, the acquisition of comprehensive functional information and direct correlation with ultrastructural details within a 3-dimensional reference space will become routine. The prospects for volume electron microscopy are therefore bright, and the stage is set for a challenging and exciting future.

The Dystonia-associated Protein TorsinA Modulates Synaptic Vesicle Recycling

The loss of a glutamic acid residue in the AAA-ATPase (ATPases associated with diverse cellular activities) torsinA is responsible for most cases of early onset autosomal dominant primary dystonia. In this study, we found that snapin, which binds SNAP-25 (synaptosome-associated protein of 25,000 Da) and enhances the association of the SNARE complex with synaptotagmin, is an interacting partner for both wild type and mutant torsinA. Snapin co-localized with endogenous torsinA on dense core granules in PC12 cells and was recruited to perinuclear inclusions containing mutant ΔE-torsinA in neuroblastoma SH-SY5Y cells. In view of these observations, synaptic vesicle recycling was analyzed using the lipophilic dye FM1-43 and an antibody directed against an intravesicular epitope of synaptotagmin I. We found that overexpression of wild type torsinA negatively affects synaptic vesicle endocytosis. Conversely, overexpression of ΔE-torsinA in neuroblastoma cells increases FM1-43 uptake. Knockdown of snapin and/or torsinA using small interfering RNAs had a similar inhibitory effect on the exo-endocytic process. In addition, down-regulation of torsinA causes the persistence of synaptotagmin I on the plasma membrane, which closely resembles the effect observed by the overexpression of the ΔE-torsinA mutant. Altogether, these findings suggest that torsinA plays a role together with snapin in regulated exocytosis and that ΔE-torsinA exerts its pathological effects through a loss of function mechanism. This may affect neuronal uptake of neurotransmitters, such as dopamine, playing a role in the development of dystonic movements.

Cell surface organization of stress-inducible proteins ULBP and MICA that stimulate human NK cells and T cells via NKG2D.

Cell surface proteins major histocompatibility complex (MHC) class I–related chain A (MICA) and UL16-binding proteins (ULBP) 1, 2, and 3 are up-regulated upon infection or tumor transformation and can activate human natural killer (NK) cells. Patches of cross-linked raft resident ganglioside GM1 colocalized with ULBP1, 2, 3, or MICA, but not CD45. Thus, ULBPs and MICA are expressed in lipid rafts at the cell surface. Western blotting revealed that glycosylphosphatidylinositol (GPI)-anchored ULBP3 but not transmembrane MICA, MHC class I protein, or transferrin receptor, accumulated in detergent-resistant membranes containing GM1. Thus, MICA may have a weaker association with lipid rafts than ULBP3, yet both proteins accumulate at an activating human NK cell immune synapse. Target cell lipid rafts marked by green fluorescent protein–tagged GPI also accumulate with ULBP3 at some synapses. Electron microscopy reveals constitutive clusters of ULBP at the cell surface. Regarding a specific molecular basis for the organization of these proteins, ULBP1, 2, and 3 and MICA are lipid modified. ULBP1, 2, and 3 are GPI anchored, and we demonstrate here that MICA is S-acylated. Finally, expression of a truncated form of MICA that lacks the putative site for S-acylation and the cytoplasmic tail can be expressed at the cell surface, but is unable to activate NK cells.

Asymmetric Segregation of Polarized Antigen on B Cell Division Shapes Presentation Capacity

Antigen Polarity in B Cell Differentiation Communication received through cell contact is critical for the differentiation of specialized effector cell populations during the immune response. For example, B lymphocytes acquire antigen that they present to helper T lymphocytes. T lymphocytes, in turn, provide key differentiation signals to B lymphocytes. In order to learn more about this process, Thaunat et al. (p. 475; see the Perspective by Dustin and Meyer-Hermann) used multiphoton microscopy and imaging flow cytometry to visualize the localization of antigen in B lymphocytes during an immune response. Antigen acquired by B lymphocytes exhibited a polarized distribution that was sustained over several rounds of cell division. This produced a population of activated B lymphocytes that contained very low levels of antigen. Daughter cells that received more antigen were better able to stimulate T cells. Because cues received through T lymphocyte interactions are likely to influence B lymphocyte fate decisions, unequal distribution of antigen in dividing B lymphocytes may influence their differentiation. Antigen distribution across activated B cells influences B-T lymphocyte interactions. During the activation of humoral immune responses, B cells acquire antigen for subsequent presentation to cognate T cells. Here we show that after mouse B cells accumulate antigen, it is maintained in a polarized distribution for extended periods in vivo. Using high-throughput imaging flow cytometry, we observed that this polarization is preserved during B cell division, promoting asymmetric antigen segregation among progeny. Antigen inheritance correlates with the ability of progeny to activate T cells: Daughter cells receiving larger antigen stores exhibit a prolonged capacity to present antigen, which renders them more effective in competing for T cell help. The generation of progeny with differential capacities for antigen presentation may have implications for somatic hypermutation and class switching during affinity maturation and as B cells commit to effector cell fates.

Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4‐mediated Dll4 expression requires a combination of integrins in vitro and integrin β1 in vivo. We conclude that appropriate laminin/integrin‐induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.