EHBP1 Is Critically Involved in the Dendritic Arbor Formation and Is Coupled to Factors Promoting Actin Filament Formation.

The coordinated action of a plethora of factors is required for the organization and dynamics of membranous structures critically underlying the development and function of cells, organs, and organisms. The evolutionary acquisition of additional amino acid motifs allows for expansion and/or specification of protein functions. We identify a thus far unrecognized motif specific for chordata EHBP1 proteins and demonstrate that this motif is critically required for interaction with syndapin I, an F-BAR domain-containing, membrane-shaping protein predominantly expressed in neurons. Gain-of-function and loss-of-function studies in rat primary hippocampal neurons (of mixed sexes) unraveled that EHBP1 has an important role in neuromorphogenesis. Surprisingly, our analyses uncovered that this newly identified function of EHBP1 did not require the domain responsible for Rab GTPase binding but was strictly dependent on EHBP1's syndapin I binding interface and on the presence of syndapin I in the developing neurons. These findings were underscored by temporally and spatially remarkable overlapping dynamics of EHBP1 and syndapin I at nascent dendritic branch sites. In addition, rescue experiments demonstrated the necessity of two additional EHBP1 domains for dendritic arborization, the C2 and CH domains. Importantly, the additionally uncovered critical involvement of the actin nucleator Cobl in EHBP1 functions suggested that not only static association with F-actin via EHBP1's CH domain is important for dendritic arbor formation but also actin nucleation. Syndapin interactions organize ternary protein complexes composed of EHBP1, syndapin I, and Cobl, and our functional data show that only together these factors give rise to proper cell shape during neuronal development.

Inositol hexakisphosphate primes syndapin I/PACSIN 1 activation in endocytosis.

Endocytosis is controlled by a well-orchestrated molecular machinery, where the individual players as well as their precise interactions are not fully understood. We now show that syndapin I/PACSIN 1 is expressed in pancreatic ß cells and that its knockdown abrogates ß cell endocytosis leading to disturbed plasma membrane protein homeostasis, as exemplified by an elevated density of L-type Ca2+ channels. Intriguingly, inositol hexakisphosphate (InsP6) activates casein kinase 2 (CK2) that phosphorylates syndapin I/PACSIN 1, thereby promoting interactions between syndapin I/PACSIN 1 and neural Wiskott-Aldrich syndrome protein (N-WASP) and driving ß cell endocytosis. Dominant-negative interference with endogenous syndapin I/PACSIN 1 protein complexes, by overexpression of the syndapin I/PACSIN 1 SH3 domain, decreases InsP6-stimulated endocytosis. InsP6 thus promotes syndapin I/PACSIN 1 priming by CK2-dependent phosphorylation, which endows the syndapin I/PACSIN 1 SH3 domain with the capability to interact with the endocytic machinery and thereby initiate endocytosis, as exemplified in ß cells.

Defining the protein and lipid constituents of tubular recycling endosomes.

Once internalized, receptors reach the sorting endosome and are either targeted for degradation or recycled to the plasma membrane, a process mediated at least in part by tubular recycling endosomes (TREs). TREs may be efficient for sorting owing to the ratio of large surface membrane area to luminal volume; following receptor segregation, TRE fission likely releases receptor-laden tubules and vesicles for recycling. Despite the importance of TRE networks for recycling, these unique structures remain poorly understood, and unresolved questions relate to their lipid and protein composition and biogenesis. Our previous studies have depicted the endocytic protein MICAL-L1 as an essential TRE constituent, and newer studies show a similar localization for the GTP-binding protein Rab10. We demonstrate that TREs are enriched in both phosphatidic acid (PA) and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), supporting the idea of MICAL-L1 recruitment by PA and Rab10 recruitment via PI(4,5)P2. Using siRNA knock-down, we demonstrate that Rab10-marked TREs remain prominent in cells upon MICAL-L1 or Syndapin2 depletion. However, depletion of Rab10 or its interaction partner, EHBP1, led to loss of MICAL-L1-marked TREs. We next used phospholipase D inhibitors to decrease PA synthesis, acutely disrupt TREs, and enable monitoring of TRE regeneration after inhibitor washout. Rab10 depletion prevented TRE regeneration, whereas MICAL-L1 knock-down did not. It is surprising that EHBP1 depletion did not affect TRE regeneration under these conditions. Overall, our study supports a primary role for Rab10 and the requirement for PA and PI(4,5)P2 in TRE biogenesis and regeneration, with Rab10 likely linking the sorting endosome to motor proteins and the microtubule network.

F-BAR domain protein Syndapin regulates actomyosin dynamics during apical cap remodeling in syncytial Drosophila embryos.

Branched actin networks driven by Arp2/3 interact with actomyosin filaments in processes such as cell migration. Similar interactions occur in the syncytial Drosophila blastoderm embryo where expansion of apical caps by Arp2/3-driven actin polymerization occurs in interphase, and cap buckling at contact edges by Myosin II to form furrows takes place in metaphase. Here, we study the role of Syndapin (Synd), an F-BAR domain-containing protein, in apical cap remodeling prior to furrow extension. We found that depletion of synd resulted in larger apical caps. Super-resolution and TIRF microscopy showed that control embryos had long apical actin protrusions in caps during interphase and short protrusions during metaphase, whereas synd depletion led to formation of sustained long protrusions, even during metaphase. Loss of Arp2/3 function in synd mutants partly reverted defects in apical cap expansion and protrusion remodeling. Myosin II levels were decreased in synd mutants, an observation consistent with the expanded cap phenotype previously reported for Myosin II mutant embryos. We propose that Synd function limits branching activity during cap expansion and affects Myosin II distribution in order to bring about a transition in actin remodeling activity from apical cap expansion to lateral furrow extension.

Regulation of caveolae through cholesterol-depletion-dependent tubulation mediated by PACSIN2.

The membrane-shaping ability of PACSIN2 (also known as syndapin II), which is mediated by its F-BAR domain, has been shown to be essential for caveolar morphogenesis, presumably through the shaping of the caveolar neck. Caveolar membranes contain abundant cholesterol. However, the role of cholesterol in PACSIN2-mediated membrane deformation remains unclear. Here, we show that the binding of PACSIN2 to the membrane can be negatively regulated by cholesterol. We prepared reconstituted membranes based on the lipid composition of caveolae. The reconstituted membrane with cholesterol had a weaker affinity for the F-BAR domain of PACSIN2 than a membrane without cholesterol. Consistent with this, upon depletion of cholesterol from the plasma membrane, PACSIN2 localized at tubules that had caveolin-1 at their tips, suggesting that cholesterol inhibits membrane tubulation mediated by PACSIN2. The tubules induced by PACSIN2 could be representative of an intermediate of caveolae endocytosis. Consistent with this, the removal of caveolae from the plasma membrane upon cholesterol depletion was diminished in the PACSIN2-deficient cells. These data suggest that PACSIN2-mediated caveolae internalization is dependent on the amount of cholesterol, providing a mechanism for cholesterol-dependent regulation of caveolae.This article has an associated First Person interview with the first author of the paper.

Syndapin constricts microvillar necks to form a united rhabdomere in Drosophila photoreceptors.

Drosophila photoreceptors develop from polarized epithelial cells that have apical and basolateral membranes. During morphogenesis, the apical membranes subdivide into a united bundle of photosensory microvilli (rhabdomeres) and a surrounding supporting membrane (stalk). By EMS-induced mutagenesis screening, we found that the F-Bin/Amphiphysin/Rvs (F-BAR) protein syndapin is essential for apical membrane segregation. The analysis of the super-resolution microscopy, STORM and the electron microscopy suggest that syndapin localizes to the neck of the microvilli at the base of the rhabdomere. Syndapin and moesin are required to constrict the neck of the microvilli to organize the membrane architecture at the base of the rhabdomere, to exclude the stalk membrane. Simultaneous loss of syndapin along with the microvilli adhesion molecule chaoptin significantly enhanced the disruption of stalk-rhabdomere segregation. However, loss of the factors involving endocytosis do not interfere. These results indicated syndapin is most likely functioning through its membrane curvature properties, and not through endocytic processes for stalk-rhabdomere segregation. Elucidation of the mechanism of this unconventional domain formation will provide novel insights into the field of cell biology.

A Novel Glycine Receptor Variant with Startle Disease Affects Syndapin I and Glycinergic Inhibition.

Glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the adult human spinal cord and brainstem. Hereditary mutations to GlyRs can lead to the rare, but potentially fatal, neuromotor disorder hyperekplexia. Most mutations located in the large intracellular domain (TM3-4 loop) of the GlyRα1 impair surface expression levels of the receptors. The novel GLRA1 mutation P366L, located in the TM3-4 loop, showed normal surface expression but reduced chloride currents, and accelerated whole-cell desensitization observed in whole-cell recordings. At the single-channel level, we observed reduced unitary conductance accompanied by spontaneous opening events in the absence of extracellular glycine. Using peptide microarrays and tandem MS-based analysis methods, we show that the proline-rich stretch surrounding P366 mediates binding to syndapin I, an F-BAR domain protein involved in membrane remodeling. The disruption of the noncanonical Src homology 3 recognition motif by P366L reduces syndapin I binding. These data suggest that the GlyRα1 subunit interacts with intracellular binding partners and may therefore play a role in receptor trafficking or synaptic anchoring, a function thus far only ascribed to the GlyRß subunit. Hence, the P366L GlyRα1 variant exhibits a unique set of properties that cumulatively affect GlyR functionality and thus might explain the neuropathological mechanism underlying hyperekplexia in the mutant carriers. P366L is the first dominant GLRA1 mutation identified within the GlyRα1 TM3-4 loop that affects GlyR physiology without altering protein expression at the whole-cell and surface levels.SIGNIFICANCE STATEMENT We show that the intracellular domain of the inhibitory glycine receptor α1 subunit contributes to trafficking and synaptic anchoring. A proline-rich stretch in this receptor domain forms a noncanonical recognition motif important for the interaction with syndapin I (PACSIN1). The disruption of this motif, as present in a human patient with hyperekplexia led to impaired syndapin I binding. Functional analysis revealed that the altered proline-rich stretch determines several functional physiological parameters of the ion channel (e.g., faster whole-cell desensitization) reduced unitary conductance and spontaneous opening events. Thus, the proline-rich stretch from the glycine receptor α1 subunit represents a multifunctional intracellular protein motif.

Syndapin I Loss-of-Function in Mice Leads to Schizophrenia-Like Symptoms.

Schizophrenia is associated with cognitive and behavioral dysfunctions thought to reflect imbalances in neurotransmission systems. Recent screenings suggested that lack of (functional) syndapin I (PACSIN1) may be linked to schizophrenia. We therefore studied syndapin I KO mice to address the suggested causal relationship to schizophrenia and to analyze associated molecular, cellular, and neurophysiological defects. Syndapin I knockout (KO) mice developed schizophrenia-related behaviors, such as hyperactivity, reduced anxiety, reduced response to social novelty, and an exaggerated novel object response and exhibited defects in dendritic arborization in the cortex. Neuromorphogenic deficits were also observed for a schizophrenia-associated syndapin I mutant in cultured neurons and coincided with a lack of syndapin I-mediated membrane recruitment of cytoskeletal effectors. Syndapin I KO furthermore caused glutamatergic hypofunctions. Syndapin I regulated both AMPAR and NMDAR availabilities at synapses during basal synaptic activity and during synaptic plasticity-particularly striking were a complete lack of long-term potentiation and defects in long-term depression in syndapin I KO mice. These synaptic plasticity defects coincided with alterations of postsynaptic actin dynamics, synaptic GluA1 clustering, and GluA1 mobility. Both GluA1 and GluA2 were not appropriately internalized. Summarized, syndapin I KO led to schizophrenia-like behavior, and our analyses uncovered associated molecular and cellular mechanisms.

The role of membrane-shaping BAR domain proteins in caveolar invagination: from mechanistic insights to pathophysiological consequences.

The formation of caveolae, bulb-shaped plasma membrane invaginations, requires the coordinated action of distinct lipid-interacting and -shaping proteins. The interdependence of caveolar structure and function has evoked substantial scientific interest given the association of human diseases with caveolar dysfunction. Model systems deficient of core components of caveolae, caveolins or cavins, did not allow for an explicit attribution of observed functional defects to the requirement of caveolar invagination as they lack both invaginated caveolae and caveolin proteins. Knockdown studies in cultured cells and recent knockout studies in mice identified an additional family of membrane-shaping proteins crucial for caveolar formation, syndapins (PACSINs) - BAR domain superfamily proteins characterized by crescent-shaped membrane binding interfaces recognizing and inducing distinct curved membrane topologies. Importantly, syndapin loss-of-function resulted exclusively in impairment of caveolar invagination without a reduction in caveolin or cavin at the plasma membrane, thereby allowing the specific role of the caveolar invagination to be unveiled. Muscle cells of syndapin III KO mice showed severe reductions of caveolae reminiscent of human caveolinopathies and were more vulnerable to membrane damage upon changes in membrane tensions. Consistent with the lack of syndapin III-dependent invaginated caveolae providing mechanoprotection by releasing membrane reservoirs through caveolar flattening, physical exercise of syndapin III KO mice resulted in pathological defects reminiscent of the clinical symptoms of human myopathies associated with caveolin 3 mutation suggesting that the ability of muscular caveolae to respond to mechanical forces is a key physiological process.

PICK1 interacts with PACSIN to regulate AMPA receptor internalization and cerebellar long-term depression.

The dynamic trafficking of AMPA receptors (AMPARs) into and out of synapses is crucial for synaptic transmission, plasticity, learning, and memory. The protein interacting with C-kinase 1 (PICK1) directly interacts with GluA2/3 subunits of the AMPARs. Although the role of PICK1 in regulating AMPAR trafficking and multiple forms of synaptic plasticity is known, the exact molecular mechanisms underlying this process remain unclear. Here, we report a unique interaction between PICK1 and all three members of the protein kinase C and casein kinase II substrate in neurons (PACSIN) family and show that they form a complex with AMPARs. Our results reveal that knockdown of the neuronal-specific protein, PACSIN1, leads to a significant reduction in AMPAR internalization following the activation of NMDA receptors in hippocampal neurons. The interaction between PICK1 and PACSIN1 is regulated by PACSIN1 phosphorylation within the variable region and is required for AMPAR endocytosis. Similarly, the binding of PICK1 to the ubiquitously expressed PACSIN2 is also regulated by the homologous phosphorylation sites within the PACSIN2-variable region. Genetic deletion of PACSIN2, which is highly expressed in Purkinje cells, eliminates cerebellar long-term depression. This deficit can be fully rescued by overexpressing wild-type PACSIN2, but not by a PACSIN2 phosphomimetic mutant, which does not bind PICK1 efficiently. Taken together, our data demonstrate that the interaction of PICK1 and PACSIN is required for the activity-dependent internalization of AMPARs and for the expression of long-term depression in the cerebellum.

Proteomic analysis of glycine receptor ß subunit (GlyRß)-interacting proteins: evidence for syndapin I regulating synaptic glycine receptors.

Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord and brainstem. They are clustered at inhibitory postsynapses via a tight interaction of their ß subunits (GlyRß) with the scaffolding protein gephyrin. In an attempt to isolate additional proteins interacting with GlyRß, we performed pulldown experiments with rat brain extracts using a glutathione S-transferase fusion protein encompassing amino acids 378-455 of the large intracellular loop of GlyRß as bait. This identified syndapin I (SdpI) as a novel interaction partner of GlyRß that coimmunoprecipitates with native GlyRs from brainstem extracts. Both SdpI and SdpII bound efficiently to the intracellular loop of GlyRß in vitro and colocalized with GlyRß upon coexpression in COS-7 cells. The SdpI-binding site was mapped to a proline-rich sequence of 22 amino acids within the intracellular loop of GlyRß. Deletion and point mutation analysis disclosed that SdpI binding to GlyRß is Src homology 3 domain-dependent. In cultured rat spinal cord neurons, SdpI immunoreactivity was found to partially colocalize with marker proteins of inhibitory and excitatory synapses. When SdpI was acutely knocked down in cultured spinal cord neurons by viral miRNA expression, postsynaptic GlyR clusters were significantly reduced in both size and number. Similar changes in GlyR cluster properties were found in spinal cultures from SdpI-deficient mice. Our results are consistent with a role of SdpI in the trafficking and/or cytoskeletal anchoring of synaptic GlyRs.

Syndapin 3 modulates fusion pore expansion in mouse neuroendocrine chromaffin cells.

Adrenal neuroendocrine chromaffin cells receive excitatory synaptic input from the sympathetic nervous system and secrete hormones into the peripheral circulation. Under basal sympathetic tone, modest amounts of freely soluble catecholamine are selectively released through a restricted fusion pore formed between the secretory granule and the plasma membrane. Upon activation of the sympathoadrenal stress reflex, elevated stimulation drives fusion pore expansion, resulting in increased catecholamine secretion and facilitating release of copackaged peptide hormones. Thus regulated expansion of the secretory fusion pore is a control point for differential hormone release of the sympathoadrenal stress response. Previous work has shown that syndapin 1 deletion alters transmitter release and that the dynamin 1-syndapin 1 interaction is necessary for coupled endocytosis in neurons. Dynamin has also been shown to be involved in regulation of fusion pore expansion in neuroendocrine chromaffin cells through an activity-dependent association with syndapin. However, it is not known which syndapin isoform(s) contributes to pore dynamics in neuroendocrine cells. Nor is it known at what stage of the secretion process dynamin and syndapin associate to modulate pore expansion. Here we investigate the expression and localization of syndapin isoforms and determine which are involved in mediating fusion pore expansion. We show that all syndapin isoforms are expressed in the adrenal medulla. Mutation of the SH3 dynamin-binding domain of all syndapin isoforms shows that fusion pore expansion and catecholamine release are limited specifically by mutation of syndapin 3. The mutation also disrupts targeting of syndapin 3 to the cell periphery. Syndapin 3 exists in a persistent colocalized state with dynamin 1.

Distinct Bin/Amphiphysin/Rvs (BAR) family proteins may assemble on the same tubule to regulate membrane organization in vivo.

Intracellular membrane tubules play a crucial role in diverse cellular processes, and their regulation is facilitated by Bin-Amphiphysin-Rvs (BAR) domain-containing proteins. This study investigates the roles of Drosophila ICA69 (dICA69) (an N-BAR protein) and Drosophila CIP4 (dCIP4) (an F-BAR protein), focusing on their impact on in vivo membrane tubule organization. In contrast to the prevailing models of BAR-domain protein function, we observed colocalization of endogenous dICA69 with dCIP4-induced tubules, indicating their potential recruitment for tubule formation and maintenance. Moreover, actin-regulatory proteins such as Wasp, SCAR, and Arp2/3 were recruited at the site of CIP4-induced tubule formation. An earlier study indicated that F-BAR proteins spontaneously segregate from the N-BAR domain proteins during membrane tubule formation. In contrast, our observation supports a model in which different BAR-domain family members can associate with the same tubule and cooperate to fine-tune the tubule width, possibly by recruiting actin modulators during the generation of tubules. Our data suggests that cooperative activities of distinct BAR-domain family proteins may determine the length and width of the membrane tubule in vivo.

The active zone protein CLA-1 (Clarinet) bridges two subsynaptic domains to regulate presynaptic sorting of ATG-9.

In neuronal synapses, autophagosome biogenesis is coupled with the activity-dependent synaptic vesicle cycle via ATG-9. How vesicles containing ATG-9 are sorted at the presynapse is unknown. We performed forward genetic screens at single synapses of C. elegans neurons for mutants that disrupt ATG-9 presynaptic localization, and identified the long isoform of the active zone protein CLA-1 (Clarinet; CLA-1 L). We find that disrupting CLA-1 L results in abnormal accumulation of ATG-9-containing vesicles enriched with clathrin. The adaptor protein complexes and proteins at the periactive zone genetically interact with CLA-1 L in ATG-9 sorting. Moreover, the phenotype of the ATG-9 protein in cla-1(L) mutants was not observed for integral synaptic vesicle proteins, suggesting distinct mechanisms that regulate sorting of ATG-9-containing vesicles and synaptic vesicles. Our findings reveal novel roles for active zone proteins in the sorting of ATG-9 and in presynaptic macroautophagy/autophagy.

Phosphorylation of PACSIN2 at S313 Regulates Podocyte Architecture in Coordination with N-WASP.

Changes in the dynamic architecture of podocytes, the glomerular epithelial cells, lead to kidney dysfunction. Previous studies on protein kinase C and casein kinase 2 substrates in neurons 2 (PACSIN2), a known regulator of endocytosis and cytoskeletal organization, reveal a connection between PACSIN2 and kidney pathogenesis. Here, we show that the phosphorylation of PACSIN2 at serine 313 (S313) is increased in the glomeruli of rats with diabetic kidney disease. We found that phosphorylation at S313 is associated with kidney dysfunction and increased free fatty acids rather than with high glucose and diabetes alone. Phosphorylation of PACSIN2 emerged as a dynamic process that fine-tunes cell morphology and cytoskeletal arrangement, in cooperation with the regulator of the actin cytoskeleton, Neural Wiskott-Aldrich syndrome protein (N-WASP). PACSIN2 phosphorylation decreased N-WASP degradation while N-WASP inhibition triggered PACSIN2 phosphorylation at S313. Functionally, pS313-PACSIN2 regulated actin cytoskeleton rearrangement depending on the type of cell injury and the signaling pathways involved. Collectively, this study indicates that N-WASP induces phosphorylation of PACSIN2 at S313, which serves as a mechanism whereby cells regulate active actin-related processes. The dynamic phosphorylation of S313 is needed to regulate cytoskeletal reorganization.

Syndapin and GTPase RAP-1 control endocytic recycling via RHO-1 and non-muscle myosin II.

After endocytosis, many plasma membrane components are recycled via membrane tubules that emerge from early endosomes to form recycling endosomes, eventually leading to their return to the plasma membrane. We previously showed that Syndapin/PACSIN-family protein SDPN-1 is required in vivo for basolateral endocytic recycling in the C. elegans intestine. Here, we document an interaction between the SDPN-1 SH3 domain and a target sequence in PXF-1/PDZ-GEF1/RAPGEF2, a known exchange factor for Rap-GTPases. We found that endogenous mutations engineered into the SDPN-1 SH3 domain, or its binding site in the PXF-1 protein, interfere with recycling in vivo, as does the loss of the PXF-1 target RAP-1. In some contexts, Rap-GTPases negatively regulate RhoA activity, suggesting a potential for Syndapin to regulate RhoA. Our results indicate that in the C. elegans intestine, RHO-1/RhoA is enriched on SDPN-1- and RAP-1-positive endosomes, and the loss of SDPN-1 or RAP-1 elevates RHO-1(GTP) levels on intestinal endosomes. Furthermore, we found that depletion of RHO-1 suppressed sdpn-1 mutant recycling defects, indicating that control of RHO-1 activity is a key mechanism by which SDPN-1 acts to promote endocytic recycling. RHO-1/RhoA is well known for controlling actomyosin contraction cycles, although little is known about the effects of non-muscle myosin II on endosomes. Our analysis found that non-muscle myosin II is enriched on SDPN-1-positive endosomes, with two non-muscle myosin II heavy-chain isoforms acting in apparent opposition. Depletion of nmy-2 inhibited recycling like sdpn-1 mutants, whereas depletion of nmy-1 suppressed sdpn-1 mutant recycling defects, indicating that actomyosin contractility controls recycling endosome function.

PACSIN proteins in vivo: Roles in development and physiology.

Protein kinase C and casein kinase substrate in neurons (PACSINs), or syndapins (synaptic dynamin-associated proteins), are a family of proteins involved in the regulation of cell cytoskeleton, intracellular trafficking and signalling. Over the last twenty years, PACSINs have been mostly studied in the in vitro and ex vivo settings, and only in the last decade reports on their function in vivo have emerged. We first summarize the identification, structure and cellular functions of PACSINs, and then focus on the relevance of PACSINs in vivo. During development in various model organisms, PACSINs participate in diverse processes, such as neural crest cell development, gastrulation, laterality development and neuromuscular junction formation. In mouse, PACSIN2 regulates angiogenesis during retinal development and in human, PACSIN2 associates with monosomy and embryonic implantation. In adulthood, PACSIN1 has been extensively studied in the brain and shown to regulate neuromorphogenesis, receptor trafficking and synaptic plasticity. Several genetic studies suggest a role for PACSIN1 in the development of schizophrenia, which is also supported by the phenotype of mice depleted of PACSIN1. PACSIN2 plays an essential role in the maintenance of intestinal homeostasis and participates in kidney repair processes after injury. PACSIN3 is abundant in muscle tissue and necessary for caveolar biogenesis to create membrane reservoirs, thus controlling muscle function, and has been linked to certain genetic muscular disorders. The above examples illustrate the importance of PACSINs in diverse physiological or tissue repair processes in various organs, and associations to diseases when their functions are disturbed.

Syndapin-2 mediated transcytosis of amyloid-ß across the blood-brain barrier.

A deficient transport of amyloid-ß across the blood-brain barrier, and its diminished clearance from the brain, contribute to neurodegenerative and vascular pathologies, such as Alzheimer's disease and cerebral amyloid angiopathy, respectively. At the blood-brain barrier, amyloid-ß efflux transport is associated with the low-density lipoprotein receptor-related protein 1. However, the precise mechanisms governing amyloid-ß transport across the blood-brain barrier, in health and disease, remain to be fully understood. Recent evidence indicates that the low-density lipoprotein receptor-related protein 1 transcytosis occurs through a tubulation-mediated mechanism stabilized by syndapin-2. Here, we show that syndapin-2 is associated with amyloid-ß clearance via low-density lipoprotein receptor-related protein 1 across the blood-brain barrier. We further demonstrate that risk factors for Alzheimer's disease, amyloid-ß expression and ageing, are associated with a decline in the native expression of syndapin-2 within the brain endothelium. Our data reveals that syndapin-2-mediated pathway, and its balance with the endosomal sorting, are important for amyloid-ß clearance proposing a measure to evaluate Alzheimer's disease and ageing, as well as a target for counteracting amyloid-ß build-up. Moreover, we provide evidence for the impact of the avidity of amyloid-ß assemblies in their trafficking across the brain endothelium and in low-density lipoprotein receptor-related protein 1 expression levels, which may affect the overall clearance of amyloid-ß across the blood-brain barrier.

Dynamin is primed at endocytic sites for ultrafast endocytosis.

Dynamin mediates fission of vesicles from the plasma membrane during endocytosis. Typically, dynamin is recruited from the cytosol to endocytic sites, requiring seconds to tens of seconds. However, ultrafast endocytosis in neurons internalizes vesicles as quickly as 50 ms during synaptic vesicle recycling. Here, we demonstrate that Dynamin 1 is pre-recruited to endocytic sites for ultrafast endocytosis. Specifically, Dynamin 1xA, a splice variant of Dynamin 1, interacts with Syndapin 1 to form molecular condensates on the plasma membrane. Single-particle tracking of Dynamin 1xA molecules confirms the liquid-like property of condensates in vivo. When Dynamin 1xA is mutated to disrupt its interaction with Syndapin 1, the condensates do not form, and consequently, ultrafast endocytosis slows down by 100-fold. Mechanistically, Syndapin 1 acts as an adaptor by binding the plasma membrane and stores Dynamin 1xA at endocytic sites. This cache bypasses the recruitment step and accelerates endocytosis at synapses.

Functional interdependence of the actin nucleator Cobl and Cobl-like in dendritic arbor development.

Local actin filament formation is indispensable for development of the dendritic arbor of neurons. We show that, surprisingly, the action of single actin filament-promoting factors was insufficient for powering dendritogenesis. Instead, this required the actin nucleator Cobl and its only evolutionary distant ancestor Cobl-like acting interdependently. This coordination between Cobl-like and Cobl was achieved by physical linkage by syndapins. Syndapin I formed nanodomains at convex plasma membrane areas at the base of protrusive structures and interacted with three motifs in Cobl-like, one of which was Ca2+/calmodulin-regulated. Consistently, syndapin I, Cobl-like's newly identified N terminal calmodulin-binding site and the single Ca2+/calmodulin-responsive syndapin-binding motif all were critical for Cobl-like's functions. In dendritic arbor development, local Ca2+/CaM-controlled actin dynamics thus relies on regulated and physically coordinated interactions of different F-actin formation-promoting factors and only together they have the power to bring about the sophisticated neuronal morphologies required for neuronal network formation in mammals.