Open questions: missing pieces from the immunological jigsaw puzzle.

AbstractFirst paragraph (this article has no abstract) There is nothing more frustrating than reaching the end of a jigsaw puzzle to find that some of the pieces are missing. There are certainly areas of immunology where the same problem applies and despite major advances there are some pieces of the jigsaw that are still missing after many years. When I was asked to think of some of the questions that remain unanswered in the cell biology of the immune system, three leapt to mind. One concerns antigen presentation, another cytotoxic T lymphocyte (CTL)- or natural killer (NK)-cell-mediated killing, and the third a mechanism of apoptosis. Although seemingly disparate, each one of these boils down to a question of how proteins cross membranes to reach the cytoplasm.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Endocytosing the death sentence

A series of recent studies have suggested that endocytosis of the mannose-6-phosphate receptor (MPR)* might play a critical role in delivering the death signal to cells targeted for destruction by the immune system (for review see Barry and Bleackley, 2002). These studies have raised a number of controversial issues regarding the trafficking of proteins from the plasma membrane of the target cell to their substrates in the cytosol. In this issue, Trapani and colleagues examine the death of cells in which endocytosis of the MPR is blocked and show that the death signal is delivered effectively in the absence of MPR endocytosis (Trapani et al., 2002, this issue). How then is the death sentence delivered

Localization of endogenous STX11 & Munc18-2 in CTL

Cytotoxic T-lymphocytes (CTL) kill their targets by cytolytic granule secretion at the immunological synapse. The Sec/Munc protein, Munc18-2, and its binding partner Syntaxin 11 (STX11) are both required for granule secretion, with mutations in either leading to the primary immunodeficiency, Familial Haemophagocytic Lymphohistiocytosis (FHL4 and 5). Understanding how Munc18-2 and STX11 function in CTL has been hampered by not knowing the endogenous localization of these proteins. Using a novel FHL5 Munc18-2 mutation that results in loss of protein, cytotoxicity and degranulation together with CTL from an FHL4 patient lacking STX11, enabled us to localize endogenous STX11 and Munc18-2 in CTL. Munc18-2 localized predominantly to cytolytic granules with low levels associated with the plasma membrane where STX11 localized. Importantly, while Munc18-2 localization is unaffected by the absence of STX11 in FHL4 CTL, STX11 is lost from the plasma membrane in FHL5 CTL lacking Munc18-2. These findings support a role for Munc18-2 in chaperoning STX11 to the plasma membrane where the final fusion events involved in secretion occur.his work was funded by grants from the Wellcome Trust [075880], [100140] and [097024/B/11/Z] (G.M.G); a Daimler Benz scholarship (Y.H.); the Kendal-Dixon Fund (Y.H.); the Cambridge European Trust (Y.H.); King’s College (Cambridge) (Y.H.); the National Institute for Health Research Biomedical Research Center (G.M.G); Antonio Pinzino – Associazione per la Ricerca sulle Sindromi Emofagocitiche (M.A.); Associazione Italiana Ricerca istiocitosi (M.A.); and Italian Ministry of Health, Progetti dioe ricerca finalizzata 2008, Bando Malattie Rare RF-TOS-2008-1219488 (M.A.).This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1111/tra.1233

Phospholipids: Pulling Back the Actin Curtain for Granule Delivery to the Immune Synapse

Phosphoinositides, together with the phospholipids phosphatidylserine and phosphatidic acid, are important components of the plasma membrane acting as second messengers that, with diacylglycerol, regulate a diverse range of signaling events converting extracellular changes into cellular responses. Local changes in their distribution and membrane charge on the inner leaflet of the plasma membrane play important roles in immune cell function. Here we discuss their distribution and regulators highlighting the importance of membrane changes across the immune synapse on the cytoskeleton and the impact on the function of cytotoxic T lymphocytes

Galaxy Halo Masses from Galaxy-Galaxy Lensing

We present measurements of the extended dark halo profiles of bright early type galaxies at redshifts 0.1 to 0.9 obtained via galaxy-galaxy lensing analysis of images taken at the CFHT using the UH8K CCD mosaic camera. Six half degree fields were observed for a total of 2 hours each in I and V, resulting in catalogs containing ~20 000 galaxies per field. We used V-I color and I magnitude to select bright early type galaxies as the lens galaxies, yielding a sample of massive lenses with fairly well determined redshifts and absolute magnitudes M ~ M_* \pm 1. We paired these with faint galaxies lying at angular distances 20" to 60", corresponding to physical radii of 26 to 77 kpc (z = 0.1) and 105 to 315 kpc (z = 0.9), and computed the mean tangential shear of the faint galaxies. The shear falls off with radius roughly as expected for flat rotation curve halos. The shear values were weighted in proportion to the square root of the luminosity of the lens galaxy. Our results give a value for the average mean rotation velocity of an L_* galaxy halo at r~50-200 kpc of v_* = 238^{+27}_{-30} km per sec for a flat lambda (Omega_m0 = 0.3, Omega_l0 = 0.7) cosmology (v_* = 269^{+34}_{-39} km per sec for Einstein-de Sitter), and with little evidence for evolution with redshift. We compare to halo masses measured by other groups/techniques. We find a mass-to-light ratio of ~121\pm28h(r/100 kpc) and these halos constitute Omega ~0.04 \pm 0.01(r/100 kpc) of closure density. (abridged)Comment: Accepted for publication in ApJ (minor modifications) - 32 pages, 11 figs, 5 table

Centriole polarisation to the immunological synapse directs secretion from cytolytic cells of both the innate and adaptive immune systems.

BACKGROUND: Cytolytic cells of the immune system destroy pathogen-infected cells by polarised exocytosis of secretory lysosomes containing the pore-forming protein perforin. Precise delivery of this lethal hit is essential to ensuring that only the target cell is destroyed. In cytotoxic T lymphocytes (CTLs), this is accomplished by an unusual movement of the centrosome to contact the plasma membrane at the centre of the immunological synapse formed between killer and target cells. Secretory lysosomes are directed towards the centrosome along microtubules and delivered precisely to the point of target cell recognition within the immunological synapse, identified by the centrosome. We asked whether this mechanism of directing secretory lysosome release is unique to CTL or whether natural killer (NK) and invariant NKT (iNKT) cytolytic cells of the innate immune system use a similar mechanism to focus perforin-bearing lysosome release. RESULTS: NK cells were conjugated with B-cell targets lacking major histocompatibility complex class I 721.221 cells, and iNKT cells were conjugated with glycolipid-pulsed CD1-bearing targets, then prepared for thin-section electron microscopy. High-resolution electron micrographs of the immunological synapse formed between NK and iNKT cytolytic cells with their targets revealed that in both NK and iNKT cells, the centrioles could be found associated (or 'docked') with the plasma membrane within the immunological synapse. Secretory clefts were visible within the synapses formed by both NK and iNKT cells, and secretory lysosomes were polarised along microtubules leading towards the docked centrosome. The Golgi apparatus and recycling endosomes were also polarised towards the centrosome at the plasma membrane within the synapse. CONCLUSIONS: These results reveal that, like CTLs of the adaptive immune system, the centrosomes of NK and iNKT cells (cytolytic cells of the innate immune system) direct secretory lysosomes to the immunological synapse. Morphologically, the overall structure of the immunological synapses formed by NK and iNKT cells are very similar to those formed by CTLs, with both exocytic and endocytic organelles polarised towards the centrosome at the plasma membrane, which forms a focal point for exocytosis and endocytosis within the immunological synapse. We conclude that centrosomal polarisation provides a rapid, responsive and precise mechanism for secretory lysosome delivery to the immunological synapse in CTLs, NK cells and iNKT cells.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Mother Centriole Distal Appendages Mediate Centrosome Docking at the Immunological Synapse and Reveal Mechanistic Parallels with Ciliogenesis.

Cytotoxic T lymphocytes (CTLs) are highly effective serial killers capable of destroying virally infected and cancerous targets by polarized release from secretory lysosomes. Upon target contact, the CTL centrosome rapidly moves to the immunological synapse, focusing microtubule-directed release at this point [1-3]. Striking similarities have been noted between centrosome polarization at the synapse and basal body docking during ciliogenesis [1, 4-8], suggesting that CTL centrosomes might dock with the plasma membrane during killing, in a manner analogous to primary cilia formation [1, 4]. However, questions remain regarding the extent and function of centrosome polarization at the synapse, and recent reports have challenged its role [9, 10]. Here, we use high-resolution transmission electron microscopy (TEM) tomography analysis to show that, as in ciliogenesis, the distal appendages of the CTL mother centriole contact the plasma membrane directly during synapse formation. This is functionally important as small interfering RNA (siRNA) targeting of the distal appendage protein, Cep83, required for membrane contact during ciliogenesis [11], impairs CTL secretion. Furthermore, the regulatory proteins CP110 and Cep97, which must dissociate from the mother centriole to allow cilia formation [12], remain associated with the mother centriole in CTLs, and neither axoneme nor transition zone ciliary structures form. Moreover, complete centrosome docking can occur in proliferating CTLs with multiple centriole pairs. Thus, in CTLs, centrosomes dock transiently with the membrane, within the cell cycle and without progression into ciliogenesis. We propose that this transient centrosome docking without cilia formation is important for CTLs to deliver rapid, repeated polarized secretion directed by the centrosome.We thank the Wellcome Trust for funding to GMG (075880) and CIMR (100140).This is the final version of the article. It was first available from Elsevier via http://dx.doi.org/10.1016/j.cub.2015.10.02

Cortical actin recovery at the immunological synapse leads to termination of lytic granule secretion in cytotoxic T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) eliminate virally infected cells through directed secretion of specialized lytic granules. Because a single CTL can kill multiple targets, degranulation must be tightly regulated. However, how CTLs regulate the termination of granule secretion remains unclear. Previous work demonstrated that centralized actin reduction at the immune synapse precedes degranulation. Using a combination of live confocal, total internal reflection fluorescence, and superresolution microscopy, we now show that, after granule fusion, actin recovers at the synapse and no further secretion is observed. Depolymerization of actin led to resumed granule secretion, suggesting that recovered actin acts as a barrier preventing sustained degranulation. Furthermore, RAB27a-deficient CTLs, which do not secrete cytotoxic granules, failed to recover actin at the synapse, suggesting that RAB27a-mediated granule secretion is required for actin recovery. Finally, we show that both actin clearance and recovery correlated with synaptic phosphatidylinositol 4,5-bisphosphate (PIP2) and that alterations in PIP2 at the immunological synapse regulate cortical actin in CTLs, providing a potential mechanism through which CTLs control cortical actin density. Our work provides insight into actin-related mechanisms regulating CTL secretion that may facilitate serial killing during immune responses