Projects Peter van der Sluijs


Short description of research
The primary objective of the group is to define the molecular principles for regulating plasma membrane plasticity. This housekeeping process is conserved in all eukaryotic cells and to some extent even in prokaryotic organisms, testifying to the essential role in the normal execution of a large variety of cellular functions. Examples of which include cytokinesis, cell polarity, cell motility, cytotoxicity of immune cells, information transfer between neurons, and development. We concentrate on those pathways that control plasticity via rapid and selective mobilization of proteins and membrane from intracellular stores. A growing number of human diseases is known to be caused by mutations in genes required for membrane transport, while acquired perturbations in aging individuals is increasingly being recognized as source for loss of quality of life. The two themes of the group are:
- degranulation of secretory lysosomes in cytolytic immune response
- endosome recycling during synaptic plasticity
Our research combines methods of immunology, genetics, biochemistry, molecular and cell biology. We also use confocal microscopy, live cell imaging (spinning disc microscopy and total internal reflection fluorescence microscopy), and immuno electronmicroscopy.


Degranulation of secretory lysosomes in cytolytic immune response

Natural Killer (NK) cells and cytotoxic T lymphocytes (CTL) are critical for immune responses against virus infections and cell transformation. The cytotoxic function is exerted through recognition of target cells, followed by regulated exocytosis of granzymes and perforin that causes apoptosis of the antigen presenting cell (Figure 1).


Figure 1. Model for CTL polarization, degranulation, and cytotoxicity. For details see text.

The effector molecules are stored in lytic granules, otherwise known as secretory lysosomes. The efficient removal of target cells via the granule-dependent pathway also serves an important function in homeostasis of lymphocytes. Genetic defects affecting cytotoxicity cause persistent release of cytokines, including IFN-γ, IL-6, IL-18 and TNF, and uncontrolled expansion of CD8 T lymphocytes and life-threatening macrophage activation syndrome.
After binding an antigen presenting cell, secretory lysosomes relocate along microtubules to the microtubule organizing center that polarizes towards the target cell. The centrosome and associated secretory lysosomes translocate together to the contact patch between effector and target cell, where docking of the centrosome delivers secretory lysosomes for fusion. This so called immunological synapse comprises a large, concentrically organized supramolecular complex of which cell adhesion molecules make up the peripheral zone while signaling proteins localize to the central core, where secretory lysososomes dock and fuse. In a proteomics screen for proteins regulating these processes we used the GTPase rab27a as bait and discovered munc13-4. Munc13-4 is a multidomain protein that like rab27a is essential for regulated release of secretory lysosomes in CTLs and NK cells. Mutations in RAB27a or MUNC13-4 cause severe human diseases Griscelli Syndrome 2 (GS2), and Familial Hemophagocytic Lymphohistiocytosis type 3 (FHL3), respectively.

The molecular principles and signals underlying the directed membrane trafficking during secretory lysosome degranulation are not clear, and controversial with respect to the functional relationship between rab27s and munc13-4. Knockouts of rab27a/b and munc13-4 produce different secretion phenotypes in platelets. In CTLs and NK cells, rab27a and munc13-4 do not localize to lytic granules unless the cells are 'activated'. We recently identified a noncanonical rab27 binding region in munc13-4 that is required and sufficient for rab27a binding (Fig. 2).


Figure 2. Model of 3D structure of munc13-4 C2A domain.
The model was made on the basis of the alignment of the C2A domain of munc13-4 with the C2B domain of rat munc13-1, whose experimental 3D structure (pdb 3kwu) has been solved. The limits of the 240-284 segment, which includes the β-strands βA to βC, are shown. Critical residues for rab27a binding are aa 280-285. Loops which were not modeled are symbolized with dashed lines. Amino acids of the Ca2+-binding site are shown together with bound Ca2+ ions (green spheres).










We made critical point mutants in this motif that were severely impaired in rab27 binding. In collaboration with the group of Geneviève de Saint Basile (Hôpital Necker-Enfants Malades, 75015, Paris) we introduced these point mutants in CTLs isolated from FHL3 patients and found that they mutants (in contrast to wild type munc13-4) could not rescue degranulation. This requirement of the munc13-4 rab27 complex is a general property of immune cells since the mutants also failed to complement in a mast cell degranulation assay that we recently developed for the molecular analysis of new FHL3 patient mutants discovered in UMC Utrecht (collaboration with Marielle E. van Gijn, Kiki Tesselaar, Lisette van de Corput). Live imaging of mast cells using total internal reflection fluoresence microscopy (collaboration with Paul van Bergen en Henegouwen, UU) revealed an essential function of munc13-4 rab27a complex in tethering secretory lysosomes to the plasma membrane. Secretory lysosomes containing wild type munc3-4 loose mobility and become tethered at the plasma membrane in activated cells in cells (Fig. 3), while those expressing munc13-4 mutants that do not bind rab27, fail to do so (Fig. 4). Present research efforts are directed towards understanding how munc13-4 exerts its function, and deciphering the signaling route from immune receptor to munc13-4 and rab27 complex.


Figure 3. Immune receptor signaling corrals secretory lysosomes at plasma membrane.
TIRFM view of YFP-munc13-4 in resting and activated RBL-2H3 cells expressing Cherry-rab27a and YFP-munc13-4. Red trajectories represent tracks of individual granules.


Figure 4. Munc13-4 rab27 is required for docking of secretory lysosomes at plasma membrane.
MSD vs Δtime plots of the tracks analyzed per cell (n=15) in resting (blue) or activated (red) Cherry-rab27a RBL-2H3 cells co-expressing YFP-munc13-4, orYFP-munc13-4(FQL>AAA) or YFP-munc13-4 Δ(280-285) that do not bind rab27. Black lines indicate the slope of the first 8s.

Endosome recycling during synaptic plasticity

Remodeling of synapses is a fundamental mechanism for information storage and processing in the brain. Endosomal pathways play a central role in synapse formation and plasticity and a popular model holds that recycling endosomes in dendrites provide the local intracellular pool of postsynaptic receptors for long-term potentiation (LTP), a cellular model for learning and memory formation. However, we are far from a complete understanding how endocytic receptor sorting and recycling is organized and coordinated in dendrites. Especially, the molecular mechanisms that couple specific endosomal trafficking routes during LTP are poorly understood. The importance of these processes for synaptic function has been well documented for AMPA-type glutamate receptors (AMPAR) that represent the major excitatory neurotransmitter receptors. Redistribution of AMPAR in and out of the synapse has emerged as an important mechanism for synaptic plasticity and information storage in the brain. Increased delivery of AMPARs to the postsynaptic membrane leads to LTP, while net removal of AMPARs by internalization from the surface seems to underlie long-term depression (LTD).

The small GTPases rab4, rab5, rab11 are directly responsible for AMPA receptor trafficking and delivery. Rabs act as binary switches that in their active form recruit/stabilize downstream effector networks and thereby function as organizers of discrete membrane microdomains. Rab5 controls transport to sorting endosomes, whereas rab4 and rab11 regulate endosomal recycling back to the plasma membrane (Fig. 5).


Figure-5 Figure 5. Distribution of rab proteins in endosomal system.

The communication and transport between sequentially organized rab domains is thought to be mediated by proteins that are 'shared' by both domains. Bivalent effectors, such as rabenosyn-5 can connect proximal rab5 and distal rab4 domains on early endosomes. How recycling endosomal domains are coupled is not well understood. Within the context of a long term collaboration with the group of Casper Hoogenraad (ErasmusMC, Rotterdam, now UU), we recently identified GRASP-1 as a neuron-specific rab4 effector that is present on recycling endosomes and can connect rab4 and rab11 domains. This link between the two recycling endosomal domains is important for normal synaptic function, since knock-down of GRASP-1 interferes with AMPAR recycling, synaptic plasticity (Fig. 6) and maintenance of spine morphology (Fig. 7).



Figure-6Figure 6. GRASP-1 regulates synaptic plasticity.
Neurons expressing GFP (to mark the dendrite) and either pSuper or pSuper-GRASP-1-shRNA#2, were stimulated with glycine (200 mM, 3 min), and then imaged for >30 min after glycine stimulation. Arrows indicated spine formation. Closed and open arrow heads spine growth and stable protrusions, respectively.

Figure-7Figure 7. GRASP-1 is required for the maintenance of dendritic spines. Representative high magnification images of dendrites of hippocampal neurons co-transfected at DIV13 for 4 days with β-galactosidase (to mark the dendrites), and either pSuper, pSuper-GRASP-1-shRNA#2, GRASP-1-shRNA#2 and GFP-GRASP-1*, Rab4S22N, and labeled with anti-β-galactosidase.

GRASP-1 is associated together with rab4 and syntaxin 13 on tubulovesicular recycling endosomes in neurons (Fig. 8). GRASP-1 not only colocalizes with these proteins, but also directly binds to rab4 and syntaxin 13 at the same time. The N-terminus of GRASP-1 interacts with rab4 while the C-terminus binds to syntaxin 13. Because syntaxin 13 is known to associate with the rab11 domain, we propose that the GRASP1-syntaxin 13 complex forms a molecular 'bridge' between the rab4 and rab11 domains on recycling endosomes (Fig. 9). Most likely one of the rab11 effector molecules is engaged in interactions with either syntaxin 13 or GRASP-1 on the recycling endosomal membrane. The precise mechanistic aspects and dynamic properties of such an endosomal recycling "bridge" are now being worked out in the group.


Figure-8Figure 8. GRASP-1, rab4 and syntaxin 13 colocalize on recycling endosomes.
Immunogold EM of hippocampal neurons labeled with 10 nm protein A gold for Rab4 and with 15 nm protein A gold for GRASP-1 (A), with 10 nm protein A gold for syntaxin 13 and with 15 nm protein A gold for GRASP-1 (B), with 10 nm protein A gold for syntaxin 13 and with 15 nm protein A gold for rab4 (C), or with 15 nm protein A gold for GRASP-1, with 5 nm protein gold for syntaxin 13, and with 10 nm protein A gold for rab4 (D). Arrow denotes tubular endosomal membrane to which GRASP-1, syntaxin 13 and rab4 localized. EE indicates early endosomes and scale bar is 100 nm.


Figure 9. Model for the role of GRASP-1 in endosome recycling.
Endosomes can be viewed as mosaic distribution of rab4, rab5 and rab11 domains that dynamically interact via effector proteins and SNAREs. The rab5 domain allows entry into the early/sorting endosome, whereas the rab4 and rab11 domains contain the machinery that is necessary for sorting and recycling membranes and receptors back to the plasma membrane. A) GRASP-1 binds to rab4 and syntaxin 13 and couples rab4 and rab11 recycling endosomes. The complex formed between GRASP-1 and t-SNARE syntaxin 13 might mediate fusion between rab4 and rab11 endosomes. B) Absence of GRASP-1 interferes with complex formation at the recycling step, causing cargo accumulation in early endosomes, impairment of receptor expression and changes in spine morphology. C) Overexpression of GRASP-1 leads to recruitment of syntaxin 13 and strongly couples rab4 and rab11 domains, causing accumulation of internalizes receptors in recycling endosomes.