SNARE Protein Mimicry by an Intracellular Bacterium

Many intracellular pathogens rely on host cell membrane compartments for their survival. The strategies they have developed to subvert intracellular trafficking are often unknown, and SNARE proteins, which are essential for membrane fusion, are possible targets. The obligate intracellular bacteria Chlamydia replicate within an intracellular vacuole, termed an inclusion. A large family of bacterial proteins is inserted in the inclusion membrane, and the role of these inclusion proteins is mostly unknown. Here we identify SNARE-like motifs in the inclusion protein IncA, which are conserved among most Chlamydia species. We show that IncA can bind directly to several host SNARE proteins. A subset of SNAREs is specifically recruited to the immediate vicinity of the inclusion membrane, and their accumulation is reduced around inclusions that lack IncA, demonstrating that IncA plays a predominant role in SNARE recruitment. However, interaction with the SNARE machinery is probably not restricted to IncA as at least another inclusion protein shows similarities with SNARE motifs and can interact with SNAREs. We modelled IncA's association with host SNAREs. The analysis of intermolecular contacts showed that the IncA SNARE-like motif can make specific interactions with host SNARE motifs similar to those found in a bona fide SNARE complex. Moreover, point mutations in the central layer of IncA SNARE-like motifs resulted in the loss of binding to host SNAREs. Altogether, our data demonstrate for the first time mimicry of the SNARE motif by a bacterium

VAMP7 modulates ciliary biogenesis in kidney cells

Epithelial cells elaborate specialized domains that have distinct protein and lipid compositions, including the apical and basolateral surfaces and primary cilia. Maintaining the identity of these domains is required for proper cell function, and requires the efficient and selective SNARE-mediated fusion of vesicles containing newly synthesized and recycling proteins with the proper target membrane. Multiple pathways exist to deliver newly synthesized proteins to the apical surface of kidney cells, and the post-Golgi SNAREs, or VAMPs, involved in these distinct pathways have not been identified. VAMP7 has been implicated in apical protein delivery in other cell types, and we hypothesized that this SNARE would have differential effects on the trafficking of apical proteins known to take distinct routes to the apical surface in kidney cells. VAMP7 expressed in polarized Madin Darby canine kidney cells colocalized primarily with LAMP2-positive compartments, and siRNA-mediated knockdown modulated lysosome size, consistent with the known function of VAMP7 in lysosomal delivery. Surprisingly, VAMP7 knockdown had no effect on apical delivery of numerous cargoes tested, but did decrease the length and frequency of primary cilia. Additionally, VAMP7 knockdown disrupted cystogenesis in cells grown in a three-dimensional basement membrane matrix. The effects of VAMP7 depletion on ciliogenesis and cystogenesis are not directly linked to the disruption of lysosomal function, as cilia lengths and cyst morphology were unaffected in an MDCK lysosomal storage disorder model. Together, our data suggest that VAMP7 plays an essential role in ciliogenesis and lumen formation. To our knowledge, this is the first study implicating an R-SNARE in ciliogenesis and cystogenesis. © 2014 Szalinski et al

Secretion of Novel SEL1L Endogenous Variants Is Promoted by ER Stress/UPR via Endosomes and Shed Vesicles in Human Cancer Cells

We describe here two novel endogenous variants of the human endoplasmic reticulum (ER) cargo receptor SEL1LA, designated p38 and p28. Biochemical and RNA interference studies in tumorigenic and non-tumorigenic cells indicate that p38 and p28 are N-terminal, ER-anchorless and more stable relative to the canonical transmembrane SEL1LA. P38 is expressed and constitutively secreted, with increase after ER stress, in the KMS11 myeloma line and in the breast cancer lines MCF7 and SKBr3, but not in the non-tumorigenic breast epithelial MCF10A line. P28 is detected only in the poorly differentiated SKBr3 cell line, where it is secreted after ER stress. Consistently with the presence of p38 and p28 in culture media, morphological studies of SKBr3 and KMS11 cells detect N-terminal SEL1L immunolabeling in secretory/degradative compartments and extracellularly-released membrane vesicles. Our findings suggest that the two new SEL1L variants are engaged in endosomal trafficking and secretion via vesicles, which could contribute to relieve ER stress in tumorigenic cells. P38 and p28 could therefore be relevant as diagnostic markers and/or therapeutic targets in cancer

Emergence and development of gut motility in the chicken embryo.

The gastrointestinal tract transports the food bolus by peristalsis. Gut motility starts at an early age in the developing embryo, well before it is required for nutrition of the organism. We present a comprehensive kinematic study of the emergence and physiological development of gut motility in all regions of the lower digestive tract of the chicken embryo from embryonic days E5 through E9. We characterized motility emergence time, propagation patterns, speed, frequency and amplitude of peristalsis waves. We found that the emergence of an uninterrupted circular ring of smooth muscle correlated with the appearance of propagative contractile waves, at E6 in the hindgut and midgut, and at E9 in the caecal appendix. We show that peristalsis at these stages is critically dependent on calcium and is not mediated by neurons as gut motility is insensitive to tetrodotoxin and takes place in the hindgut in the absence of neurons. We further demonstrate that motility also matures in ex-vivo organ culture. We compare our results to existing literature on zebrafish, mouse and human motility development, and discuss their chronological relationship with other major developmental events occurring in the chicken embryonic gut at these stages. Our work sets a baseline for further investigations of motility development in this important animal model

Emergence and development of gut motility in the chicken embryo - Fig 7

<p>a) Evolution of jejunal contractile wave frequency and amplitude of E4 guts (n = 3) kept up to 7 days in culture. Circled data points indicate that the full time-lapse video corresponding to this condition is available as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172511#pone.0172511.s009" target="_blank">S9</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172511#pone.0172511.s010" target="_blank">S10</a> Videos. b) Immunohistochemical staining of neurons (green, β-III tubulin) and smooth muscle (red, α-actin) in native E4 midgut (left) and E4 midgut kept 4 days in culture (right). The brightfield image is overlaid in grey. For the native E4 midgut, the epithelium stains weakly and non-specifically to Alexa488.</p

Representative motility patterns at E5, E6 and E7 (S2–S4 Videos).

<p>At E5, the motiligram is deduced from the sole beating region of the gut which is the distal jejunum (dashed box). At later stages, the motiligram is placed right below the region of the gut from which it was derived; spatial scales are the same for still images of the gut and motiligrams. The main anatomical segments of the gut are indicated at E6, HG: hindgut, IL: ileum, JEJ: jejunum, umb: umbilicus.</p

Analysis of embryonic gut motility.

<p>a) Stereomicroscope single frame from a time-lapse stack showing an E9 jejunum resting on an Anodisc membrane. Black arrows: three constrictions travelling from right to left (abanal waves). b) Motiligram (= kymograph) computed from the region-of-interest boxed by red dashed lines in a). Aborally and abanally propagating waves are indicated, as well as wave annihilation (“a”) and nucleation events (“n”); the speed <i>v</i> of a wave is deduced from the tilt angle <i>α</i> by <i>v</i> = tan <i>α</i>. c) Magnified view of a constriction (E8 jejunum). The constriction is not symmetric (dashed red lines): the edge of the gut ahead of the propagating constriction has a steeper incline than the edge of the gut in back of the constriction. I: smooth muscle and myenteric plexus region; II: mucosa, epithelium and lumen region. The deformation (= strain) due to the constriction is concentrated in region II; the thickness of region I does not change during the constriction. The full time-lapse video is available as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172511#pone.0172511.s001" target="_blank">S1 Video</a>.</p

Effect of TTX and CoCl₂ at E7 (a,b) and E9 (c,d).

<p>a,c) Representative motiligrams in control conditions, after the addition of TTX 1 μM and after the addition of CoCl₂ 1 mM. b,d) Changes in frequency, speed and amplitude after addition of TTX or CoCl₂. Number of samples: E7 n = 6, E9 n = 4. The speed <i>v</i> is related to the tilt angle <i>α</i> by <i>v</i> = tan <i>α</i>. A star indicates a statistically significant difference (p<0.05, Mann-Whitney two-tailed test). In d) we separated the immediate effects of CoCl₂ from its effect after 20 min application; we also indicate when the amplitude vanishes (“a.v.”) or fades after addition of CoCl₂. The wave speed in the caecal appendix immediately following CoCl₂ application was in the range 70–350 μm/sec.</p

Immunohistochemical staining of neurons (green, β-III tubulin) and smooth muscle (red, α-actin) in the hindgut, jejunum (pre-umbilical midgut) and ileum (IL)—caeca (CC) region from E5 to E9, transverse sections.

<p>The brightfield image is overlaid in grey. The scale bar is the same for all sections. The enteric nervous system appears as rings of neurons located around the circular smooth muscle layer; concentrated, non-axisymmetric patches of neurons visible on the periphery of sections E5-HG and E9-HG are the extrinsic innervation (white arrows). The epithelium is visible on the brightfield image, but sometimes stains non-specifically either to Alexa488 (e.g., E7-IL, E8-HG) or to CY3 (e.g., E8-JEJ) secondary antibody.</p