Colin M. Segovis

Cutting Edge: Syntaxin 11 Regulates Lymphocyte-Mediated Secretion and Cytotoxicity

Little is known about the regulatory roles of specific soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in cytotoxic lymphocytes. Recent information suggests that mutations in the SNARE protein syntaxin 11 result in a form of familial hemophagocytic lymphohistiocytosis (FHL). Because genetic abnormalities in key granule components (e.g., perforin) or in regulators of secretion (e.g., Munc13–4) underlie the other identified forms of FHL, we assessed whether syntaxin 11 might also serve a related regulatory role. We determined that syntaxin 11 is expressed in NK cells and activated CTLs and is located in discrete membrane-associated structures in the cytoplasm. Enhanced expression of syntaxin 11 augments the secretion and killing of tumor targets, and suppression of syntaxin 11 expression inhibits these functions. Our data identify and characterize a role for syntaxin 11 in granule exocytosis and in the generation of cell-mediated killing. These results also provide new insights on the mechanisms of hemopoietic dysregulation in FHL.

The WAVE2 complex regulates T cell receptor signaling to integrins via Abl- and CrkL–C3G-mediated activation of Rap1

WAVE2 regulates T cell receptor (TCR)–stimulated actin cytoskeletal dynamics leading to both integrin clustering and affinity maturation. Although WAVE2 mediates integrin affinity maturation by recruiting vinculin and talin to the immunological synapse in an Arp2/3-dependent manner, the mechanism by which it regulates integrin clustering is unclear. We show that the Abl tyrosine kinase associates with the WAVE2 complex and TCR ligation induces WAVE2-dependent membrane recruitment of Abl. Furthermore, we show that WAVE2 regulates TCR-mediated activation of the integrin regulatory guanosine triphosphatase Rap1 via the recruitment and activation of the CrkL–C3G exchange complex. Moreover, we demonstrate that although Abl does not regulate the recruitment of CrkL–C3G into the membrane, it does affect the tyrosine phosphorylation of C3G, which is required for its guanine nucleotide exchange factor activity toward Rap1. This signaling node regulates not only TCR-stimulated integrin clustering but also affinity maturation. These findings identify a previously unknown mechanism by which the WAVE2 complex regulates TCR signaling to Rap1 and integrin activation.

PI3K Links NKG2D Signaling to a CrkL Pathway Involved in Natural Killer Cell Adhesion, Polarity, and Granule Secretion

The NK cell-activating receptor NKG2D plays a critical role in the destruction of malignant cells, but many of the cell-signaling mechanisms governing NKG2D-mediated cellular cytotoxicity are unknown. We have identified an NKG2D-mediated signaling pathway that governs both conjugate formation and cytotoxic granule polarization. We demonstrate that an interaction between the regulatory subunit of PI3K, p85, and the adaptor protein CrkL is required for efficient NKG2D-mediated cellular cytotoxicity. We show decreased NK cell-target cell conjugate formation in NK cells treated with PI3K inhibitors or depleted of CrkL. Independent of adhesion, we find that microtubule organization center polarization toward target cells expressing the NKG2D ligand MICA or toward anti-NKG2D-coated beads is impaired in the absence of CrkL. Ab-stimulated granule release is also impaired in NK cells depleted of CrkL. Furthermore, our data indicate that the small Ras family GTPase Rap1 is activated downstream of NKG2D engagement in a PI3K- and CrkL-dependent manner and is required for conjugate formation, MTOC (microtubule organizing center) polarization, and NKG2D-dependent cellular cytotoxicity. Taken together, our data identify an NKG2D-activated signaling pathway that collectively orchestrates NK cell adhesion, cell polarization, and granule release.

If you feed them, they will come: A prospective study of the effects of complimentary food on attendance and physician attitudes at medical grand rounds at an academic medical center

BackgroundEvidence suggests that attendance at medical grand rounds at academic medical centers is waning. The present study examined whether attendance at medical grand rounds increased after providing complimentary food to attendees and also assessed attendee attitudes about complimentary food.MethodsIn this prospective, before-and-after study, attendance at medical grand rounds was monitored from September 25, 2002, to June 2, 2004, using head counts. With unrestricted industry (eg, pharmaceutical) financial support, complimentary food was provided to medical grand rounds attendees beginning June 4, 2003. Attendance was compared during the pre-complimentary food and complimentary food periods. Attitudes about the complimentary food were assessed with use of a survey administered to attendees at the conclusion of the study period.ResultsThe mean (± SD) overall attendance by head counts increased 38.4% from 184.1 ± 90.4 during the pre-complimentary food period to 254.8 ± 60.5 during the complimentary food period (P < .001). At the end of the study period, 70.1% of the attendee survey respondents indicated that they were more likely to attend grand rounds because of complimentary food, 53.6% indicated that their attendance increased as a result of complimentary food, and 53.1% indicated that their attendance would decrease if complimentary food was no longer provided. Notably, 80.3% indicated that food was not a distraction, and 81.7% disagreed that industry representatives had influence over medical grand rounds because of their financial support for the food.ConclusionProviding free food may be an effective strategy for increasing attendance at medical grand rounds.

Dynamin 2 Regulates Granule Exocytosis during NK Cell-Mediated Cytotoxicity

NK cells are innate immune cells that can eliminate their targets through granule release. In this study, we describe a specialized role for the large GTPase Dynamin 2 (Dyn2) in the regulation of these secretory events leading to cell-mediated cytotoxicity. By modulating the expression of Dyn2 using small interfering RNA or by inhibiting its activity using a pharmacological agent, we determined that Dyn2 does not regulate conjugate formation, proximal signaling, or granule polarization. In contrast, during cell-mediated killing, Dyn2 localizes with lytic granules and polarizes to the NK cell–target interface where it regulates the final fusion of lytic granules with the plasma membrane. These findings identify a novel role for Dyn2 in the exocytic events required for effective NK cell-mediated cytotoxicity.

TCP/IP Optimization over Wide Area Networks: Implications for Teleradiology

Radiology examinations are large. The advent of fast volume imaging is making that statement truer every year. PACS are based on the assumption of fast local networking and just-in-time image pull to the desktop. On the other hand, teleradiology has been developed on a push model to accommodate the challenges of moderate bandwidth, high-latency wide area networks (WANs). Our group faced the challenging task of creating a PACS environment that felt local, while pulling images across a 3,000-mile roundtrip WAN link. Initial tests showed WAN performance lagging local area network (LAN) performance by a factor of 30 times. A 16-month journey of explorations pulled the WAN value down to only 1.5 times slower than the LAN.