CHL1 Is a Selective Organizer of the Presynaptic Machinery Chaperoning the SNARE Complex

Proteins constituting the presynaptic machinery of vesicle release undergo substantial conformational changes during the process of exocytosis. While changes in the conformation make proteins vulnerable to aggregation and degradation, little is known about synaptic chaperones which counteract these processes. We show that the cell adhesion molecule CHL1 directly interacts with and regulates the activity of the synaptic chaperones Hsc70, CSP and αSGT. CHL1, Hsc70, CSP and αSGT form predominantly CHL1/Hsc70/αSGT and CHL1/CSP complexes in synapses. Among the various complexes formed by CHL1, Hsc70, CSP and αSGT, SNAP25 and VAMP2 induce chaperone activity only in CHL1/Hsc70/αSGT and CHL1/CSP complexes, respectively, indicating a remarkable selectivity of a presynaptic chaperone activity for proteins of the exocytotic machinery. In mice with genetic ablation of CHL1, chaperone activity in synapses is reduced and the machinery for synaptic vesicle exocytosis and, in particular, the SNARE complex is unable to sustain prolonged synaptic activity. Thus, we reveal a novel role for a cell adhesion molecule in selective activation of the presynaptic chaperone machinery.     

The adhesion molecule CHL1 regulates uncoating of clathrin-coated synaptic vesicles

In searching for binding partners of the intracellular domain of the immunoglobulin superfamily adhesion molecule CHL1, we identified the clathrin-uncoating ATPase Hsc70. CHL1 gene ablation resulted in reduced targeting of Hsc70 to the synaptic plasma membrane and synaptic vesicles, suggesting CHL1 as a synapse-targeting cue for Hsc70. CHL1 accumulates in presynaptic membranes and, in response to synapse activation, is targeted to synaptic vesicles by endocytosis. CHL1 deficiency or disruption of the CHL1/Hsc70 complex results in accumulation of abnormally high levels of clathrin-coated synaptic vesicles with a reduced ability to release clathrin. Generation of new clathrin-coated synaptic vesicles in an activity-dependent manner is inhibited when the CHL1/Hsc70 complex is disrupted, resulting in impaired uptake and release of FM dyes in synaptic boutons. Abnormalities in clathrin-dependent synaptic vesicle recycling may thus underlie brain malfunctions in humans and mice that carry mutations in the CHL1 gene.     

Systematic analysis of barrier-forming FG hydrogels from Xenopus nuclear pore complexes

Nuclear pore complexes (NPCs) control the traffic between cell nucleus and cytoplasm. While facilitating translocation of nuclear transport receptors (NTRs) and NTR·cargo complexes, they suppress passive passage of macromolecules ?30 kDa. Previously, we reconstituted the NPC barrier as hydrogels comprising S. cerevisiae FG domains. We now studied FG domains from 10 Xenopus nucleoporins and found that all of them form hydrogels. Related domains with low FG motif density also substantially contribute to the NPC's hydrogel mass. We characterized all these hydrogels and observed the strictest sieving effect for the Nup98‐derived hydrogel. It fully blocks entry of GFP‐sized inert objects, permits facilitated entry of the small NTR NTF2, but arrests importin β‐type NTRs at its surface. O‐GlcNAc modification of the Nup98 FG domain prevented this arrest and allowed also large NTR·cargo complexes to enter. Solid‐state NMR spectroscopy revealed that the O‐GlcNAc‐modified Nup98 gel lacks amyloid‐like β‐structures that dominate the rigid regions in the S. cerevisiae Nsp1 FG hydrogel. This suggests that FG hydrogels can assemble through different structural principles and yet acquire the same NPC‐like permeability.     

Cohesiveness tunes assembly and morphology of FG nucleoporin domain meshworks – Implications for nuclear pore permeability

Nuclear pore complexes control the exchange of macromolecules between the cytoplasm and the nucleus. A selective permeability barrier that arises from a supramolecular assembly of intrinsically unfolded nucleoporin domains rich in phenylalanine-glycine dipeptides (FG domains) fills the nuclear pore. There is increasing evidence that selective transport requires cohesive FG domain interactions. To understand the functional roles of cohesive interactions, we studied monolayers of end-grafted FG domains as a bottom-up nanoscale model system of the permeability barrier. Based on detailed physicochemical analysis of the model films and comparison of the data with polymer theory, we propose that cohesiveness is tuned to promote rapid assembly of the permeability barrier and to generate a stable and compact pore-filling meshwork with a small mesh size. Our results highlight the functional importance of weak interactions, typically a few k_BT per chain, and contribute important information to understand the mechanism of size-selective transport.     

The permeability of reconstituted nuclear pores provides direct evidence for the selective phase model

Nuclear pore complexes (NPCs) maintain a permeability barrier between the nucleus and the cytoplasm through FG-repeat-containing nucleoporins (Nups). We previously proposed a "selective phase model" in which the FG repeats interact with one another to form a sieve-like barrier that can be locally?disrupted by the binding of nuclear transport receptors (NTRs), but not by inert macromolecules, allowing selective passage of NTRs and associated cargo. Here, we provide direct evidence for this model in a physiological context. By using NPCs reconstituted from Xenopus laevis egg extracts, we show that Nup98 is essential for maintaining the permeability barrier. Specifically, the multivalent cohesion between FG repeats is required, including cohesive FG repeats close to the anchorage point to the NPC scaffold. Our data exclude alternative models that are based solely on an interaction between the FG repeats and NTRs and indicate that the barrier is formed by a sieve-like FG hydrogel.     

Small molecule induction of MSH2-dependent cell death suggests a vital role of mismatch repair proteins in cell death

Avoidance of apoptosis is one of the hallmarks of cancer development and progression. Chemotherapeutic agents aim to initiate an apoptotic response, but often fail due to dysregulation. MSH proteins are capable of recognizing cisplatin damage in DNA and participate in the initiation of cell death. We have exploited this recognition and computationally simulated a MutS homolog (MSH) "death conformation". Screening and docking experiments based on this model determined that the MSH2-dependent cell-death pathway can be induced by a small molecule without DNA damage, reserpine. Reserpine was identified via virtual screening on structures obtained from molecular dynamics as a small molecule that selectively binds a protein "death" conformation. The virtual screening predicts that this small molecule binds in the absence of DNA. Cell biology confirmed that reserpine triggers the MSH2-dependent cell-death pathway. This result supports the hypothesis that the MSH2-dependent pathway is initiated by specific protein conformational changes triggered by binding to either DNA damage or small compound molecules. These findings have multiple implications for drug discovery and cell biology. Computational modeling may be used to identify and eventually design small molecules that selectively activate particular pathways through conformational control. Molecular dynamics simulations can be used to model the biologically relevant conformations and virtual screening can then be used to select for small molecules that bind specific conformations. The ability of a small molecule to induce the cell-death pathway suggests a broader role for MMR proteins in cellular events, such as cell-death pathways, than previously suspected.