Dynamic membrane remodeling during intracellular trafficking is controlled by the intricate interplay between lipids and proteins. BAR domains are modules that participate in endocytic processes by binding and deforming the lipid bilayer. Sorting nexin 9 (SNX9), which functions in clathrin-mediated endocytosis, contains a BAR domain, however, the properties of this domain are not well understood. Here we show that SNX9 shares many properties with other BAR domain-containing proteins, such as amphiphysin and endophilin. SNX9 is able to deform the plasma membrane, as well as liposomes, into narrow tubules and recruit N-WASP and dynamin 2 to these tubules via its SH3 domain. SNX9-induced tubulation is antagonized by N-WASP and dynamin 2 while it is enhanced by perturbation of actin dynamics. However, SNX9 also has several unique properties. The tubulating activity requires the BAR and PX domains, as well as the low-complexity (LC) domain, which binds the Arp2/3 complex. SNX9 also binds to PtdIns(4)P-5-kinases via its PX domain and its tubulating activity is regulated by phosphoinositides. In addition, the kinase activity of PtdIns(4)P-5-kinases is stimulated by interaction with SNX9, suggesting a positive feedback interaction between SNX9 and PtdIns(4)P-5-kinases. These results suggest that SNX9 functions in the coordination of membrane remodeling and fission via interactions with actin-regulating proteins, endocytic proteins and PtdIns(4,5)P 2 -metabolizing enzymes.

Sorting nexin 9 (SNX9) is a member of the sorting nexin family of proteins, each of which contains a characteristic Phox homology domain. SNX9 is widely expressed and plays a role in clathrin-mediated endocytosis, but it is not known if it is present in neuronal cells. We report that SNX9 is expressed in the presynaptic compartment of cultured hippocampal neurons, where it binds to dynamin-1 and N-WASP. Overexpression of fulllength SNX9 or a C-terminal truncated version caused severe defects in synaptic vesicle endocytosis during, as well as after, stimulation. Knockdown of SNX9 with short interfering RNA also reduced synaptic vesicle endocytosis, and the W39A mutation of SNX9 abolished the inhibitory effect of SNX9 on endocytosis. Rescue experiments showed that most of the effect of SNX9 on endocytosis results from its interaction with dynamin 1, although its interaction with N-WASP contributes in some degree. We further showed that SNX9 dimerizes through its C-terminal domain, suggesting that it may interact simultaneously with dynamin 1 and N-WASP. We propose that SNX9 interacts with dynamin-1 and N-WASP in presynaptic terminals, where it links actin dynamics and synaptic vesicle endocytosis.Sorting nexin 9 (SNX9), 2 also known as SH3PX1, is a member of the sorting nexin superfamily characterized by the presence of a phospholipid-binding motif, the PX domain. Sorting nexin family proteins contribute to protein sorting in cells by their ability to bind specific lipids and to form protein-protein complexes. SNX9, initially identified as a protein interacting with the metalloproteases MDC9 and MDC15 (1), is composed of an N-terminal Src homology 3 domain, a low complexity region, a PX domain, and a C-terminal Bin/Amphiphysin/Rvs (BAR) domain (2-4). It forms a complex with dynamin-2 and regulates the recruitment of dynamin-2 to the membrane (5). It also enhances the assembly of dynamin and increases its GTPase activity (6). Other endocytic molecules, namely AP-2 (adaptor protein complex 2) and clathrin, also bind to the low complexity region of SNX9 in a cooperative manner (2). Through these interactions, SNX9 plays an important role in clathrin-mediated endocytosis in non-neuronal cells (2, 6).Dynamin is centrally involved in clathrin-mediated endocytosis (7,8). It self-assembles around the necks of invaginated clathrin-coated pits and releases vesicles from the membrane via GTP hydrolysis (9). It is composed of several domains. The N-terminal nucleotide-binding domain is responsible for GTP hydrolysis, and the C-terminal proline-rich domain (PRD) links it to several SH3 domain-containing proteins such as Grb2, amphiphysin, and endophilin (10 -12). The central pleckstrin homology domain controls its binding to membrane phospholipids (13), and a coiled-coil domain (also called the GTPase effector domain) is involved in its self-assembly and in regulating its GTPase activity.The affinity between the pleckstrin homology domain of dynamin and lipids is not high enough to translocate dynamin from the cytosol to the p...

Excitatory stimuli are known to be a potent regulator for induction of neuronal differentiation. Calbindin-D28K buffers intracellular Ca2+ and modifies synaptic functions in neurons. However, the effects of calbindin-D28K on the regulation of activity-induced neuronal differentiation and related biochemical modifications remain unsolved. In the present study, by a gain-of-function study with retroviral vector system and dicer-generated small interfering RNA (d-siRNA) to effectively knock down the expression of calbindin-D28K, we demonstrated that calbindin-D28K at a physiologically relevant level promoted neuronal differentiation and neurite outgrowth. Increase of neuronal differentiation by calbindin-D28K overexpression was concurrent with the expression of basic helix-loop-helix (bHLH) transcriptional factors, phosphorylation of calcium and calmodulin-dependent protein kinase II (CaMKII) and NeuroD at Ser(336). KN-62, a highly specific CaMKII inhibitor, blocked the up-regulation of proneural bHLH genes, p-CaMKII, and pSer(336)NeuroD. Calbindin-D28K appeared to facilitate neuronal differentiation of both fetal and adult hippocampal progenitor cells. Together, these findings establish the novel calbindin-regulated function of CaMKII and NeuroD in control of neuronal differentiation and neurite outgrowth.

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