Britta Qualmann

Temporal and spatial coordination of exocytosis and endocytosis

In secretory cells, exocytosis and compensatory endocytosis are tightly coupled membrane trafficking processes that control the surface area and composition of the plasma membrane. While exocytic and endocytic processes have been studied independently in great detail, at present there is much interest in understanding the mode of their coupling. This review discusses emerging insights into the coupling of these processes, both in the chemical synapses of neurons and in non-neuronal cells.

Neural Wiskott Aldrich Syndrome Protein (N-WASP) and the Arp2/3 complex are recruited to sites of clathrin-mediated endocytosis in cultured fibroblasts

Several findings suggest that actin-mediated motility can play a role in clathrin-mediated endocytosis but it remains unclear whether and when key proteins required for this process are recruited to endocytic sites. Here we investigate this question in live Swiss 3T3 cells using two-colour evanescent field (EF) microscopy. We find that Arp3, a component of the Arp2/3 complex, appears transiently while single clathrin-coated pits internalize. There is also additional recruitment of Neural-Wiskott Aldrich Syndrome Protein (N-WASP), a known activator of the Arp2/3 complex. Both proteins appear at about the same time as actin. We suggest that N-WASP and the Arp2/3 complex trigger actin polymerization during a late step in clathrin-mediated endocytosis, and propel clathrin-coated pits or vesicles from the plasma membrane into the cytoplasm.

The presynaptic cytomatrix of brain synapses.

Abstract. Synapses are principal sites for communication between neurons via chemical messengers called neurotransmitters. Neurotransmitters are released from presynaptic nerve terminals at the active zone, a restricted area of the cell membrane situated exactly opposite to the postsynaptic neurotransmitter reception apparatus. At the active zone neurotransmitter-containing synaptic vesicles (SVs) dock, fuse, release their content and are recycled in a strictly regulated manner. The cytoskeletal matrix at the active zone (CAZ) is thought to play an essential role in the organization of this SV cycle. Several multi-domain cytoskeleton-associated proteins, including RIM, Bassoon, Piccolo/Aczonin and Munc-13, have been identified, which are specifically localized at the active zone and thus are putative molecular components of the CAZ. This review will summarize our present knowledge about the structure and function of these CAZ-specific proteins. Moreover, we will review our present view of how the exocytotic and endocytic machineries at the site of neurotransmitter release are linked to and organized by the presynaptic cytoskeleton. Finally, we will summarize recent progress that has been made in understanding how active zones are assembled during nervous system development.

Endocytosis and the cytoskeleton.

In this review we describe the potential roles of the actin cytoskeleton in receptor-mediated endocytosis in mammalian cells and summarize the efforts of recent years in establishing a relationship between these two cellular functions. With molecules such as dynamin, syndapin, HIP1R, Abp1, synaptojanin, N-WASP, intersectin, and cortactin a set of molecular links is now available and it is likely that their further characterization will reveal the basic principles of a functional interconnection between the membrane cytoskeleton and the vesicle-budding machinery. We will therefore discuss proteins involved in endocytic clathrin coat formation and accessory factors to control and regulate coated vesicle formation but we will also focus on actin cytoskeletal components such as the Arp2/3 complex, spectrin, profilin, and motor proteins involved in actin dynamics and organization. Additionally, we will discuss how phosphoinositides, such as PI(4,5)P2, small GTPases thought to control the actin cytoskeleton, such as Rho, Rac, and Cdc42, or membrane trafficking, such as Rab GTPases and ARF proteins, and different kinases may participate in the functional connection of actin and endocytosis. We will compare the concepts and different molecular mechanisms involved in mammalian cells with yeast as well as with specialized cells, such as epithelial cells and neurons, because different model organisms often offer complementary advantages for further studies in this thriving field of current cell biological research.

Syndapins integrate N‐WASP in receptor‐mediated endocytosis

Syndapins are potential links between the cortical actin cytoskeleton and endocytosis because this family of dynamin‐associated proteins can also interact with the Arp2/3 complex activator N‐WASP. Here we provide evidence for involvement of N‐WASP interactions in receptor‐mediated endocytosis. We reveal that the observed dominant‐negative effects of N‐WASP are dependent exclusively on the proline‐rich domain, the binding interface of syndapins. Our results therefore suggest that syndapins integrate N‐WASP functions in endocytosis. Both proteins co‐localize in neuronal cells. Consistent with a crucial role for syndapins in endocytic uptake, co‐overexpression of syndapins rescued the endocytosis block caused by N‐WASP. An in vivo reconstitution of the syndapin–N‐WASP interaction at cellular membranes triggered local actin polymerization. Depletion of endogenous N‐WASP by sequestering it to mitochondria or by introducing anti‐N‐WASP antibodies impaired endocytosis. Our data suggest that syndapins may act as important coordinators of N‐WASP and dynamin functions during the different steps of receptor‐mediated endocytosis and that local actin polymerization induced by syndapin–N‐WASP interactions may be a mechanism supporting clathrin‐coated vesicle detachment and movement away from the plasma membrane.

Regulation of endocytic traffic by Rho GTPases.

The members of the Rho subfamily of small GTPases are key regulators of the actin cytoskeleton. However, recent studies have provided evidence for multiple additional roles for these signalling proteins in controlling endocytic traffic. Here we review our current understanding of Rho GTPase action within the endocytic pathway and examine the potential points of convergence with the more established, actin-based functions of these signalling proteins.

Regulation of N-WASP and the Arp2/3 Complex by Abp1 Controls Neuronal Morphology

Polymerization and organization of actin filaments into complex superstructures is indispensable for structure and function of neuronal networks. We here report that knock down of the F-actin-binding protein Abp1, which is important for endocytosis and synaptic organization, results in changes in axon development virtually identical to Arp2/3 complex inhibition, i.e., a selective increase of axon length. Our in vitro and in vivo experiments demonstrate that Abp1 interacts directly with N-WASP, an activator of the Arp2/3 complex, and releases the autoinhibition of N-WASP in cooperation with Cdc42 and thereby promotes N-WASP-triggered Arp2/3 complex-mediated actin polymerization. In line with our mechanistical studies and the colocalization of Abp1, N-WASP and Arp2/3 at sites of actin polymerization in neurons, we reveal an essential role of Abp1 and its cooperativity with Cdc42 in N-WASP-induced rearrangements of the neuronal cytoskeleton. We furthermore show that introduction of N-WASP mutants lacking the ability to bind Abp1 or Cdc42, Arp2/3 complex inhibition, Abp1 knock down, N-WASP knock down and Arp3 knock down, all cause identical neuromorphological phenotypes. Our data thus strongly suggest that these proteins and their complex formation are important for cytoskeletal processes underlying neuronal network formation.

Complexes of syndapin II with dynamin II promote vesicle formation at the trans-Golgi network

The role of dynamin and so-called accessory proteins in endocytosis is well established. However, molecular details of the function(s) of dynamin II at the Golgi are largely unclear. We demonstrate that the ubiquitously expressed syndapin II isoform interacts with the proline-rich domain (PRD) of dynamin II through its Src-homology 3 (SH3) domain. Co-immunoprecipitation of endogenous syndapin II and dynamin II, and successful reconstitutions of such complexes at membranes in COS-7 cells, show the in vivo relevance of the interaction. Syndapin II can associate with Golgi membranes and this association increases upon Golgi exit block. Brefeldin A treatment clearly shows that the observed perinuclear localization of syndapin II co-localizing with syntaxin 6 reflects the Golgi complex and that it requires functional integrity of the Golgi. Syndapins are crucial for Golgi vesicle formation because anti-syndapin antibodies, used either in in vitro reconstitutions or in living cells, inhibited this process. Both types of assays additionally revealed the essential role of syndapin II SH3 interactions with the dynamin II PRD in vesicle formation. An excess of the syndapin SH3 domain strongly inhibited budding from Golgi membranes in vitro. Likewise, overexpression of the syndapin SH3 domain or of a dynamin II variant incapable of associating with syndapin II (dynamin IIΔPRD) impaired trafficking of vesicular stomatitis virus glycoprotein (VSVG)-GFP in vivo. By contrast, full-length syndapin II-l had no negative effect, and instead promoted VSVG-GFP export from the Golgi. Importantly, a cytosolic fraction containing endogenous syndapin-dynamin complexes was sufficient to promote vesicle formation from Golgi membranes in a syndapin-dependent manner. Thus, syndapin-dynamin complexes are crucial and sufficient to promote vesicle formation from the trans-Golgi network.

Extending the court for cortactin: from the cortex to the Golgi

Vesicle formation at the trans-Golgi network may be mechanistically more similar to endocytic vesicle formation at the plasma membrane than previously thought. Both processes share common components including the dynamin-binding protein cortactin.