regulation of polarized membrane transport by rab GTPases

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					regulation of polarized membrane transport by rab GTPases




      Peter van der Sluijs; p.vandersluijs@umcutrecht.nl
 cytotoxic T cell recognizes complex of viral peptide
        with MHC class I and kills infected cell




CTL




                                      infected cell
                                                 janeway et al , 2005
if this does not work: hemophagocytic syndrome




                                           macrophage with ingested
                                           platelets and a lymphocyte
      time course and modalities of T cell secretory responses

                                                        today:




CTL




                                                                 huse et al, 2008
proteins linked to MTOC polarization and release of
              lytic granules and cytokines



  Early TCR signaling:
  Src kinases, Zap70, Lat, SLP-76, Vav and PLC-gamma

  MTOC polarization:
  ADAP, Dynein, FMNL1, Dia1, IQGAP, Rac, Cdc42, Pyk2, Erk, HDAC6

  Lytic-granule release:
  Rab27a, AP-3, Munc13-4, Syntaxin-11, Slp1, Slp2, Paxillin, PKC-
  delta, WIP

  Synaptic cytokine secretion:
  Rab3d, Rab19, Syntaxin-4, SNAP23, WASP

  Multidirectional cytokine secretion:
  Syntaxin-6




                                                                    huse et al, 2008
  transient adhesive interactions between T cells and APCs are
             stabilized by specific antigen recognition




       CTL
CD8




                                                          janeway et al, 2005
T cells (CD4 and CD8) form compositionally and functionally distinct
                            synapses




 CD4+, stable
                                                dSMAC: actin

                                                pSMAC: adhesion

                                                cSMAC: signaling and secretion



 CD8+, transient
                  sequence of events leading to target cell killing by CTLs




collision and non-specific adhesion




re-organization of CTL architecture




release of granules at site of contact



                                                                       janeway et al, 2005
APC interaction with T cell- regulates actin cytoskeleton




                                                     billadeau et al, 2007
multiple signalling pathways are controlled by cdc42 + crosstalk
TCR-mediated MTOC polarization




                                 billadeau et al, 2007
secretory lysosomes combine features of secretory granules and lysosomes




          fibroblast            secretory cell    haematopoietic cell




                                                         clark and griffiths, 2003
haematopoietic cells and functions




                                     janeway et al , 2005
related architecture of immune receptor signaling pathways
                   in haematopoietic cells



     mast cell                                  T lymphocyte




                                Activation of
                                downstream      MAPK         integrin
                                effectors       activation   activation
intracellular distribution of rab proteins




                                             schwartz, et al. 2007
                      the rabGTPase switch



regulatory          GTP                 GDP
signals

                            GEF
                                               GTP
GDI                                                           effector
              rab                            rab
                                                              targets
REP          GDP


                            GAP


                                                     other GTPases:
                     Pi                              ran, ras, rho, ARF
rab proteins coordinate membrane transport via effectors




                                                      munro, 2005
characteristic features of rab GTPases
                two mechanisms for delivery of rabs to a membrane




- REP pathway for newly synthesized rab
- GDI pathway for re-utilization of rab
synthesis route of lipid tail that is appended to C terminus of rabs
rab27a regulates localization and exocytosis of lysosome-related organelles in
            different cell types through distinct effector proteins




                                                                      olkkonen, 2006
                 affinity purification of rab effectors


                                      B                         B

                                                               GSTrabGDP
GSH bead   GST            GSH bead                GSH bead
                                     GSTrabGTP

  A                         A             C         A               D




           SDS-PAGE


                      C                                      LC/MS-MS
                      D

                      B

                      A
               isolation of munc13-4 from spleen


silver stain        western blot




                     direct binding assay




                                                   neeft et al, 2005
munc13-4 and rab27a are highly expressed in some haematopoietic cells




                                                munc13-4

                                                rab27a


                                                actin




                                                             neeft et al, 2005
comparison of GS2 and FHL3 granule CTL phenotypes suggests
            munc13-4 acts downstream of rab27a




   normal                GS2, rab27a       FHL3, munc13-4




                                              docking of SLs,
                      no docking of SLs,      no fusion with PM
                      no fusion with PM

                                                 de saint basile et al, 2003
 mast cell degranulation assay




resting                  activated

           IgE/TNP-BSA
                                     janeway et al, 2005
    munc13-4 is a positive regulator of mast cell degranulation




1                                                       1090
     -hexosaminidase release (% of mock)




                                           150


                                           125


                                           100


                                           75


                                           50
                                                       YFP-Munc13-4   GFP-Munc13-4   GFP-Munc13-4
                                                 GFP     wild type     (543-1090)       608-611)    neeft et al, 2005
        model for munc13-1 function in neuronal synapse




                                                    synaptic vesicles




pre-synaptic neuron                                 Munc13-1



  exocytosis
                                   releasable   primed             docked
                              2+
      neurotransmitter   Ca




  post-synaptic cell
munc13 is possibly involved in assembly of
      SNARE complex at synapse




                                             brose, 2007
                          munc13-4 and rab27a colocalize on secretory lysosomes

YFP-munc13-4/CFP-rab27a
                                         p80            serotonin                   CD63




                          p80                   serotonin            CD63
                          YFP-munc13-4          YFP-munc13-4         YFP-munc13-4
                          CFP-rab27a            CFP-rab27a           CFP-rab27a


                                                                              neeft et al, 2005
                          munc13-4 localization on granules requires MHDs



            YFPmunc13-4        YFPmunc13-4(1-543)      YFPmunc13-4(543-1090)   YFPmunc13-4(283-917)
serotonin




                                             granule localization domain
                                                                                        neeft et al, 2005
    FHL3 mutant munc13-4 608-611 is mislocalized




                                         merge




GFPmunc13-4 (608-611)      serotonin




                                                   neeft et al, 2005
                        608-611
is munc13-4/rab27a complex required for secretory lysosome release?




                  how to approach the question
rab27a binding site is in N-terminus of munc13-4




              rab27BD
                                                   neeft et al, 2005
               munc13-4 truncations for determining rab27a-BD



     1                                                                                                 1090
FL             C2                                       MHD1             MHD 2             C2
         125        221                           557      671         785     893   925        1016




                                                                         rab27a binding
          A               240                                    543          +
          B                     290                              543           -
          C                           340                        543           -
          D                                 390                  543           -
          E                                       440            543          -
          G               240           290                                   +
          H               240                 340                             +


                                                                                                       maaike neeft
ala scan of aa 240-290 in munc13-4 reveals critical region for rab27 binding




                                                      maaike neeft and mark cheung
specific knock-down of munc13-4 in RBL-2H3 cells proteins




                                           edo elstak and parisa goodarzifard
                                rab27a binding mutants fail to rescue degranulation in mast cells
beta-hexosaminidase secretion




                                                      lentiviral munc13-4 construct



                                                                                                    edo elstak
lytic granule secretion at the immune synapse requires a complex of
                          rab27 an munc13-4
has the problem be solved and can we derive a unifying model for
  munc13-4 and rab27a function in secretory lysosome release?


                                                 rab11



                                    Granzyme B
                                                                        rab7
           Munc13-4                 Perforin
                                    Lamp1,2
                                                                        rab27a

                                                  Munc13-4



                      rab27a




                                                               rab27a




          mast cell                                      CTL

       compound exocytosis                     serial exocytosis
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Peter van der Sluijs; p.vandersluijs@umcutrecht.nl