Harry Mellor

Actin stress fibres.

Animal cell movement is effected through a combination of protrusive and contractile events. Non-muscle cells contain stress fibres – bundles of actomyosin that are the major mediators of cell contraction and that can be compared to the highly organised actomyosin arrays of muscle cells. Recent studies have defined regulatory mechanisms that control stress fibre formation, placing the ROCK protein kinase at the centre of a complex signalling network controlling actomyosin contractility and stress fibre assembly. As we uncover the details of stress fibre construction, it is becoming clear that different categories of stress fibres exist. Some of these structures are less suited for cell motility and more suited to static contraction. In keeping with this, many specialised contractile cell types use stress fibres to remodel tissues and extracellular matrix.

Sorting Nexin-1 Mediates Tubular Endosome-to-TGN Transport through Coincidence Sensing of High- Curvature Membranes and 3-Phosphoinositides

BACKGROUND Sorting nexins (SNXs) are phox homology (PX) domain-containing proteins thought to regulate endosomal sorting of internalized receptors. The prototypical SNX is sorting nexin-1 (SNX1), a protein that through its PX domain binds phosphatidylinositol 3-monophosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)]. SNX1 is associated with early endosomes, from where it has been proposed to regulate the degradation of internalized epidermal growth factor (EGF) receptors through modulating endosomal-to-lysosomal sorting. RESULTS We show here that SNX1 contains a BAR (Bin/Amphiphysin/Rvs) domain, a membrane binding domain that endows SNX1 with the ability to form dimers and to sense membrane curvature. We present evidence that through coincidence detection, the BAR and PX domains efficiently target SNX1 to a microdomain of the early endosome defined by high curvature and the presence of 3-phosphoinositides. In addition, we show that the BAR domain endows SNX1 with an ability to tubulate membranes in-vitro and drive the tubulation of the endosomal compartment in-vivo. Using RNA interference (RNAi), we establish that SNX1 does not play a role in EGF or transferrin receptor sorting; rather it specifically perturbs endosome-to-trans Golgi network (TGN) transport of the cation-independent mannose-6-phosphate receptor (CI-MPR). Our data support an evolutionarily conserved function for SNX1 from yeast to mammals and provide functional insight into the molecular mechanisms underlying lipid-mediated protein targeting and tubular-based protein sorting. CONCLUSIONS We conclude that through coincidence detection SNX1 associates with a microdomain of the early endosome-characterized by high membrane curvature and the presence of 3-phosphoinositides-from where it regulates tubular-based endosome-to-TGN retrieval of the CI-MPR.

Regulation of endocytic traffic by Rho family GTPases

Endocytosis is a complicated yet highly efficient process that involves the uptake and processing of cargoes, ranging from small molecules, to activated signalling receptors, to whole microorganisms. Regulation of endocytic pathways is poorly understood. Recent evidence suggests that the Rho GTPase family of signalling proteins is intimately involved in endocytic traffic, providing novel insights into the control mechanisms that govern this process.

Regulation of epidermal growth factor receptor traffic by the small GTPase RhoB

Members of the Rho family of small GTPases control cell adhesion and motility through dynamic regulation of the actin cytoskeleton. Although twelve family members have been identified, only three of these - RhoA, Rac and Cdc42 - have been studied in detail. RhoA regulates the formation of focal adhesions and the bundling of actin filaments into stress fibres. It is also involved in other cell signalling pathways including the regulation of gene expression and the generation of lipid second messengers [1] [2]. RhoA is very closely related to two other small GTPases about which much less is known: RhoB and RhoC (which are approximately 83% identical). Perhaps the most intriguing of these is RhoB. RhoA is largely cytosolic but translocates to the plasma membrane on activation. RhoB, however, is entirely localised to the cytosolic face of endocytic vesicles [3] [4]. This suggests a potential role for RhoB in regulating endocytic traffic; however, no evidence has been presented to support this. RhoA has been shown to act at the plasma membrane to regulate the clathrin-mediated internalisation of transferrin receptor [5] and of the muscarinic acetylcholine receptor [6]. We have recently demonstrated that RhoB binds the RhoA effector, PRK1 and targets it to the endosomal compartment [7]. We show here that RhoB acts through PRK1 to regulate the kinetics of epidermal growth factor receptor traffic.

Co-ordinate regulation of distinct host cell signalling pathways by multifunctional enteropathogenic Escherichia coli effector molecules

Enteropathogenic Escherichia coli (EPEC) is a major cause of paediatric diarrhoea and a model for the family of attaching and effacing (A/E) pathogens. A/E pathogens encode a type III secretion system to transfer effector proteins into host cells. The EPEC Tir effector protein acts as a receptor for the bacterial surface protein intimin and is involved in the formation of Cdc42‐independent, actin‐rich pedestal structures beneath the adhered bacteria. In this paper, we demonstrate that EPEC binding to HeLa cells also induces Tir‐independent, cytoskeletal rearrangement evidenced by the early, transient formation of filopodia‐like structures at sites of infection. Filopodia formation is dependent on expression of the EPEC Map effector molecule – a protein that targets mitochondria and induces their dysfunction. We show that Map‐induced filopodia formation is independent of mitochondrial targeting and is abolished by cellular expression of the Cdc42 inhibitory WASP‐CRIB domain, demonstrating that Map has at least two distinct functions in host cells. The transient nature of the filopodia is related to an ability of EPEC to downregulate Map‐induced cell signalling that, like pedestal formation, was dependent on both Tir and intimin proteins. The ability of Tir to downregulate filopodia was impaired by disrupting a putative GTPase‐activating protein (GAP) motif, suggesting that Tir may possess such a function, with its interaction with intimin triggering this activity. Furthermore, we also found that Map‐induced cell signalling inhibits pedestal formation, revealing that the cellular effects of Tir and Map must be co‐ordinately regu‐lated during infection. Possible implications of the multifunctional nature of EPEC effector molecules in pathogenesis are discussed.

The Phox Homology (PX) Domain-dependent, 3-Phosphoinositide-mediated Association of Sorting Nexin-1 with an Early Sorting Endosomal Compartment Is Required for Its Ability to Regulate Epidermal Growth Factor Receptor Degradation

Recent studies have shown that phox homology (PX) domains act as phosphoinositide-binding motifs. The majority of PX domains studied show binding to phosphatidylinositol 3-monophosphate (PtdIns(3)P), an association that allows the host protein to localize to membranes of the endocytic pathway. One issue, however, is whether PX domains may have alternative phosphoinositide binding specificities that could target their host protein to distinct subcellular compartments or allow their allosteric regulation by phosphoinositides other than PtdIns(3)P. It has been reported that the PX domain of sorting nexin 1 (SNX1) specifically binds phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) (Zhong, Q., Lazar, C. S., Tronchere, H., Sato, T., Meerloo, T., Yeo, M., Songyang, Z., Emr, S. D., and Gill, G. N. (2002)Proc. Natl. Acad. Sci. U. S. A. 99, 6767–6772). In the present study, we have shown that whereas SNX1 binds PtdIns(3,4,5)P3 in protein:lipid overlay assays, in liposomes-based assays, binding is observed to PtdIns(3)P and phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) but not to PtdIns(3,4,5)P3. To address the significance of PtdIns(3,4,5)P3 binding, we examined the subcellular localization of SNX1 under conditions in which plasma membrane PtdIns(3,4,5)P3 levels were significantly elevated. Under these conditions, we failed to observe association of SNX1 with this membrane. However, consistent with the binding to PtdIns(3)P and PtdIns(3,5)P2 being of more physiological significance was the observation that the association of SNX1 with an early endosomal compartment was dependent on a 3-phosphoinositide-binding PX domain and the presence of PtdIns(3)P on this compartment. Finally, we have shown that the PX domain-dependent/early endosomal association of SNX1 is important for its ability to regulate the targeting of internalized epidermal growth factor receptor for lysosomal degradation.

Cell cycle-dependent phosphorylation of the translational repressor eIF-4E binding protein-1 (4E-BP1)

A fundamental control point in the regulation of the initiation of protein synthesis is the formation of the eukaryotic initiation factor 4F (eIF-4F) complex. The formation of this complex depends upon the availability of the mRNA cap binding protein, eIF-4E, which is sequestered away from the translational machinery by the tight association of eIF-4E binding proteins (4E-BPs). Phosphorylation of 4E-BP1 is critical in causing its dissociation from eIF-4E, leaving 4E available to form translationally active eIF-4F complexes, switching on mRNA translation. In this report, we provide the first evidence that the phosphorylation of 4E-BP1 increases during mitosis and identify Ser-65 and Thr-70 as phosphorylated sites. Phosphorylation of Thr-70 has been implicated in the regulation of 4E-BP1 function, but the kinase phosphorylating this site was unknown. We show that the cyclin-dependent kinase, cdc2, phosphorylates 4E-BP1 at Thr-70 and that phosphorylation of this site is permissive for Ser-65 phosphorylation. Crucially, the increased phosphorylation of 4E-BP1 during mitosis results in its complete dissociation from eIF-4E.

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.

The novel Rho-family GTPase rif regulates coordinated actin-based membrane rearrangements.

Small GTPases of the Rho family have a critical role in controlling cell morphology, motility and adhesion through dynamic regulation of the actin cytoskeleton [1,2]. Individual Rho GTPases have been shown to regulate distinct components of the cytoskeletal architecture; RhoA stimulates the bundling of actin filaments into stress fibres [3], Rac reorganises actin to produce membrane sheets or lamellipodia [4] and Cdc42 causes the formation of thin, actin-rich surface projections called filopodia [5]. We have isolated a new Rho-family GTPase, Rif (Rho in filopodia), and shown that it represents an alternative signalling route to the generation of filopodial structures. Coordinated regulation of Rho-family GTPases can be used to generate more complicated actin rearrangements, such as those underlying cell migration [6]. In addition to inducing filopodia, Rif functions cooperatively with Cdc42 and Rac to generate additional structures, increasing the diversity of actin-based morphology.

The role of formins in filopodia formation

Filopodia are highly dynamic cell-surface protrusions used by cells to sense their external environment. At the core of the filopodium is a bundle of actin filaments. These give form to the filopodia and also drive the cycle of elongation and retraction. Recent studies have shown that two very different actin nucleating proteins control the formation of filopodial actin filaments - Arp2/3 and Formins. Although the actin filaments produced by these two nucleators have very different structures and properties, recent work has begun to piece together evidence for co-operation between Arp2/3 and formins in filopodia formation, leading to a deeper understanding of these sensory organelles.