Actin related proteins (Arps) are a highly conserved family of proteins that have extensive seque... more Actin related proteins (Arps) are a highly conserved family of proteins that have extensive sequence and structural similarity to actin. All characterized Arps are components of large multimeric complexes associated with chromatin or the cytoskeleton. In addition, the human genome encodes five conserved but largely uncharacterized ″orphan″ Arps, which appear to be mostly testis specific. Here we show that Arp7A, which has 43% sequence identity with β-actin, forms a complex with the cytoskeletal proteins Tes and Mena in the subacrosomal layer of round spermatids. The N-terminal 65 residue extension to the actin-like fold of Arp7A interacts directly with Tes. The crystal structure of the 1-65Arp7A:LIM2-3Tes:EVH1Mena complex reveals that residues 28 - 49 of Arp7A contact the LIM2-3 domains of Tes. Two alanine residues from Arp7A that occupy equivalent apolar pockets in both LIM domains as well as an intervening GPAK linker that binds the LIM2-3 junction, are critical for the Arp7A:Tes interaction. Equivalent occupied apolar pockets are also seen in the tandem LIM domain structures of LMO4 and Lhx3 bound to unrelated ligands. Our results indicate that apolar pocket interactions are a common feature of tandem LIM domain interactions, but ligand specificity is principally determined by the linker sequence.
The intracellular targeting of Ena/VASP family members is achieved via the interaction of their E... more The intracellular targeting of Ena/VASP family members is achieved via the interaction of their EVH1 domain with FPPPP sequence motifs found in a variety of cytoskeletal proteins, including lamellipodin, vinculin, and zyxin. Here we show that the LIM3 domain of Tes, which lacks the FPPPP motif, binds to the EVH1 domain of Mena, but not to those of VASP or Evl. The structure of the LIM3:EVH1 complex reveals that Tes occludes the FPPPP-binding site and competes with FPPPP-containing proteins for EVH1 binding. Structure-based gain-of-function experiments define the molecular basis for the specificity of the Tes-Mena interaction. Consistent with in vitro observations, the LIM3 domain displaces Mena, but not VASP, from the leading edge and focal adhesions. It also regulates cell migration through a Mena-dependent mechanism. Our observations identify Tes as an atypical EVH1 binding partner and a regulator specific to a single Ena/VASP family member.
Viruses are obligate intracellular parasites that are critically dependent on their hosts to repl... more Viruses are obligate intracellular parasites that are critically dependent on their hosts to replicate and generate new progeny. To achieve this goal, viruses have evolved numerous elegant strategies to subvert and utilise the different cellular machineries and processes of their unwilling hosts. Moreover, they often accomplish this feat with a surprisingly limited number of proteins. Among the different systems of the cell, the cytoskeleton is often one of the first to be hijacked as it provides a convenient transport system for viruses to reach their site of replication with relative ease. At the latter stages of their replication cycle, the cytoskeleton also provides an efficient means for newly assembled viral progeny to reach the plasma membrane and leave the infected cell. In this review we discuss how Vaccinia virus takes advantage of the microtubule and actin cytoskeletons of its host to promote the spread of infection into neighboring cells. In particular, we highlight how ...
Paradoxically, the thymidine kinase (TK) encoded by Kaposi sarcoma-associated herpesvirus (KSHV) ... more Paradoxically, the thymidine kinase (TK) encoded by Kaposi sarcoma-associated herpesvirus (KSHV) is an extremely inefficient nucleoside kinase, when compared to TKs from related herpesviruses. We now show that KSHV-TK, in contrast to HSV1-TK, associates with the actin cytoskeleton and induces extensive cell contraction followed by membrane blebbing. These dramatic changes in cell morphology depend on the auto-phosphorylation of tyrosines 65, 85 and 120 in the N-terminus of KSHV-TK. Phosphorylation of tyrosines 65/85 and 120 results in an interaction with Crk family proteins and the p85 regulatory subunit of PI3-Kinase, respectively. The interaction of Crk with KSHV-TK leads to tyrosine phoshorylation of this cellular adaptor. Auto-phosphorylation of KSHV-TK also induces a loss of FAK and paxillin from focal adhesions, resulting in activation of RhoA-ROCK signalling to myosin II and cell contraction. In the absence of FAK or paxillin, KSHV-TK has no effect on focal adhesion integrity...
Vaccinia virus, a close relative of the causative agent of smallpox, exploits actin polymerizatio... more Vaccinia virus, a close relative of the causative agent of smallpox, exploits actin polymerization to enhance its cell-to-cell spread. We show that actin-based motility of vaccinia is initiated only at the plasma membrane and remains associated with it. There must therefore be another form of cytoplasmic viral transport, from the cell centre, where the virus replicates, to the periphery. Video analysis reveals that GFP-labelled intracellular enveloped virus particles (IEVs) move from their perinuclear site of assembly to the plasma membrane on microtubules. We show that the viral membrane protein A36R, which is essential for actin-based motility of vaccinia, is also involved in microtubule-mediated movement of IEVs. We further show that conventional kinesin is recruited to IEVs via the light chain TPR repeats and is required for microtubule-based motility of the virus. Vaccinia thus sequentially exploits the microtubule and actin cytoskeletons to enhance its cell-to-cell spread.
Vasodilator-stimulated phosphoprotein (VASP) is involved in multiple actin-mediated processes, in... more Vasodilator-stimulated phosphoprotein (VASP) is involved in multiple actin-mediated processes, including regulation of serum response factor (SRF) activity. We used the SRF transcriptional assay to de®ne functional domains in VASP and to show that they coincide with those required for F-actin accumulation, as determined by a quantitative FACS assay. We identi-®ed inactive VASP mutants that can interfere both with F-actin assembly and with SRF activation by wild-type VASP. These VASP mutants also inhibit actin-based motility of Vaccinia virus and Shigellā exneri. VASP-induced F-actin accumulation and SRF activation require both functional Rho and its effector mDia, and conversely, mDia-mediated SRF activation is critically dependent on functional VASP. VASP and mDia also associate physically in vivo. These ®ndings show that VASP and mDia function cooperatively downstream of Rho to control F-actin assembly and SRF activity.
of a cobalamin-binding domain (M, = 28,000) have been grown in polyethylene glycol 6000 at pH 7.5... more of a cobalamin-binding domain (M, = 28,000) have been grown in polyethylene glycol 6000 at pH 7.5, starting from solutions of intact (M, = 133,000) cobalamin-dependent
How can Ena/VASP proteins promote actin-based movement of the intracellular pathogen Listeria or ... more How can Ena/VASP proteins promote actin-based movement of the intracellular pathogen Listeria or rapid protrusion of lamellipodia but at the same time inhibit cell translocation? A report in the May 17(th) issue of Cell now offers a possible explanation for this conundrum. Bear et al. report that Ena/VASP proteins regulate cell motility by competing with capping proteins to control actin filament length and geometry at the leading edge of cells.
The role of clathrin in pathogen entry has received much attention and has highlighted the adapta... more The role of clathrin in pathogen entry has received much attention and has highlighted the adaptability of clathrin during internalization. Recent studies have now uncovered additional roles for clathrin and have put the spotlight on its role in pathogen spread. Here, we discuss the manipulation of clathrin by pathogens, with specific attention to the processes that occur at the plasma membrane. In the majority of cases, both clathrin and the actin cytoskeleton are hijacked, so we also examine the interplay between these two systems and their role during pathogen internalization, egress and spread.
Vaccinia virus infection dramatically affects the host actin cytoskeleton by inducing disassembly... more Vaccinia virus infection dramatically affects the host actin cytoskeleton by inducing disassembly of actin stress fibres and formation of actin tails which propel the virus intra-and intercellularly. The viral factors responsible for these actin rearrangements remain unknown. Sequence analysis reveals significant homology between the vaccinia F8L ORF and the proline repeats of iActA, the protein which initiates actin tail assembly and motility in the bacterial pathogen Listeria ivanovii. We characterized the F8L gene product to examine its possible role in vaccinia rearrangements of the host actin cytoskeleton. F8L is a " 8 kDa protein expressed early during infection and is found throughout the cytoplasm, with no discernible association with viral or cellular structures. Furthermore, the F8L deletion strain, WR∆F8L, forms particles and actin tails indistinguishable from WR. Our observations demonstrate that F8L is not required for vaccinia virus morphogenesis or the actin rearrangements observed during infection.
The adaptor protein Nck has been shown to link receptor ligation to actin-based signalling in a d... more The adaptor protein Nck has been shown to link receptor ligation to actin-based signalling in a diverse range of cellular events, such as changes in cell morphology and motility. It has also been implicated in phagocytosis. However, its molecular role in controlling actin remodelling associated with phagocytic uptake remains to be clarified. Here, we show that Nck, which is recruited to phagocytic cups, is required for Fcc receptor (FccR)-but not complement receptor 3 (CR3)-induced phagocytosis. Nck recruitment in response to FccR ligation is mediated by the phosphorylation of tyrosine 282 and 298 in the ITAM motif in the cytoplasmic tail of the receptor. In the absence of FccR phosphorylation, there is also no recruitment of N-WASP or Cdc42 to phagocytic cups. Nck promotes FccR-mediated phagocytosis by recruiting N-WASP to phagocytic cups. Efficient phagocytosis, however, only occurs, if the CRIB domain of N-WASP can also interact with Cdc42. Our observations demonstrate that Nck and Cdc42 collaborate to stimulate N-WASP-dependent FccR-mediated phagocytosis.
Actin related proteins (Arps) are a highly conserved family of proteins that have extensive seque... more Actin related proteins (Arps) are a highly conserved family of proteins that have extensive sequence and structural similarity to actin. All characterized Arps are components of large multimeric complexes associated with chromatin or the cytoskeleton. In addition, the human genome encodes five conserved but largely uncharacterized ″orphan″ Arps, which appear to be mostly testis specific. Here we show that Arp7A, which has 43% sequence identity with β-actin, forms a complex with the cytoskeletal proteins Tes and Mena in the subacrosomal layer of round spermatids. The N-terminal 65 residue extension to the actin-like fold of Arp7A interacts directly with Tes. The crystal structure of the 1-65Arp7A:LIM2-3Tes:EVH1Mena complex reveals that residues 28 - 49 of Arp7A contact the LIM2-3 domains of Tes. Two alanine residues from Arp7A that occupy equivalent apolar pockets in both LIM domains as well as an intervening GPAK linker that binds the LIM2-3 junction, are critical for the Arp7A:Tes interaction. Equivalent occupied apolar pockets are also seen in the tandem LIM domain structures of LMO4 and Lhx3 bound to unrelated ligands. Our results indicate that apolar pocket interactions are a common feature of tandem LIM domain interactions, but ligand specificity is principally determined by the linker sequence.
The intracellular targeting of Ena/VASP family members is achieved via the interaction of their E... more The intracellular targeting of Ena/VASP family members is achieved via the interaction of their EVH1 domain with FPPPP sequence motifs found in a variety of cytoskeletal proteins, including lamellipodin, vinculin, and zyxin. Here we show that the LIM3 domain of Tes, which lacks the FPPPP motif, binds to the EVH1 domain of Mena, but not to those of VASP or Evl. The structure of the LIM3:EVH1 complex reveals that Tes occludes the FPPPP-binding site and competes with FPPPP-containing proteins for EVH1 binding. Structure-based gain-of-function experiments define the molecular basis for the specificity of the Tes-Mena interaction. Consistent with in vitro observations, the LIM3 domain displaces Mena, but not VASP, from the leading edge and focal adhesions. It also regulates cell migration through a Mena-dependent mechanism. Our observations identify Tes as an atypical EVH1 binding partner and a regulator specific to a single Ena/VASP family member.
Viruses are obligate intracellular parasites that are critically dependent on their hosts to repl... more Viruses are obligate intracellular parasites that are critically dependent on their hosts to replicate and generate new progeny. To achieve this goal, viruses have evolved numerous elegant strategies to subvert and utilise the different cellular machineries and processes of their unwilling hosts. Moreover, they often accomplish this feat with a surprisingly limited number of proteins. Among the different systems of the cell, the cytoskeleton is often one of the first to be hijacked as it provides a convenient transport system for viruses to reach their site of replication with relative ease. At the latter stages of their replication cycle, the cytoskeleton also provides an efficient means for newly assembled viral progeny to reach the plasma membrane and leave the infected cell. In this review we discuss how Vaccinia virus takes advantage of the microtubule and actin cytoskeletons of its host to promote the spread of infection into neighboring cells. In particular, we highlight how ...
Paradoxically, the thymidine kinase (TK) encoded by Kaposi sarcoma-associated herpesvirus (KSHV) ... more Paradoxically, the thymidine kinase (TK) encoded by Kaposi sarcoma-associated herpesvirus (KSHV) is an extremely inefficient nucleoside kinase, when compared to TKs from related herpesviruses. We now show that KSHV-TK, in contrast to HSV1-TK, associates with the actin cytoskeleton and induces extensive cell contraction followed by membrane blebbing. These dramatic changes in cell morphology depend on the auto-phosphorylation of tyrosines 65, 85 and 120 in the N-terminus of KSHV-TK. Phosphorylation of tyrosines 65/85 and 120 results in an interaction with Crk family proteins and the p85 regulatory subunit of PI3-Kinase, respectively. The interaction of Crk with KSHV-TK leads to tyrosine phoshorylation of this cellular adaptor. Auto-phosphorylation of KSHV-TK also induces a loss of FAK and paxillin from focal adhesions, resulting in activation of RhoA-ROCK signalling to myosin II and cell contraction. In the absence of FAK or paxillin, KSHV-TK has no effect on focal adhesion integrity...
Vaccinia virus, a close relative of the causative agent of smallpox, exploits actin polymerizatio... more Vaccinia virus, a close relative of the causative agent of smallpox, exploits actin polymerization to enhance its cell-to-cell spread. We show that actin-based motility of vaccinia is initiated only at the plasma membrane and remains associated with it. There must therefore be another form of cytoplasmic viral transport, from the cell centre, where the virus replicates, to the periphery. Video analysis reveals that GFP-labelled intracellular enveloped virus particles (IEVs) move from their perinuclear site of assembly to the plasma membrane on microtubules. We show that the viral membrane protein A36R, which is essential for actin-based motility of vaccinia, is also involved in microtubule-mediated movement of IEVs. We further show that conventional kinesin is recruited to IEVs via the light chain TPR repeats and is required for microtubule-based motility of the virus. Vaccinia thus sequentially exploits the microtubule and actin cytoskeletons to enhance its cell-to-cell spread.
Vasodilator-stimulated phosphoprotein (VASP) is involved in multiple actin-mediated processes, in... more Vasodilator-stimulated phosphoprotein (VASP) is involved in multiple actin-mediated processes, including regulation of serum response factor (SRF) activity. We used the SRF transcriptional assay to de®ne functional domains in VASP and to show that they coincide with those required for F-actin accumulation, as determined by a quantitative FACS assay. We identi-®ed inactive VASP mutants that can interfere both with F-actin assembly and with SRF activation by wild-type VASP. These VASP mutants also inhibit actin-based motility of Vaccinia virus and Shigellā exneri. VASP-induced F-actin accumulation and SRF activation require both functional Rho and its effector mDia, and conversely, mDia-mediated SRF activation is critically dependent on functional VASP. VASP and mDia also associate physically in vivo. These ®ndings show that VASP and mDia function cooperatively downstream of Rho to control F-actin assembly and SRF activity.
of a cobalamin-binding domain (M, = 28,000) have been grown in polyethylene glycol 6000 at pH 7.5... more of a cobalamin-binding domain (M, = 28,000) have been grown in polyethylene glycol 6000 at pH 7.5, starting from solutions of intact (M, = 133,000) cobalamin-dependent
How can Ena/VASP proteins promote actin-based movement of the intracellular pathogen Listeria or ... more How can Ena/VASP proteins promote actin-based movement of the intracellular pathogen Listeria or rapid protrusion of lamellipodia but at the same time inhibit cell translocation? A report in the May 17(th) issue of Cell now offers a possible explanation for this conundrum. Bear et al. report that Ena/VASP proteins regulate cell motility by competing with capping proteins to control actin filament length and geometry at the leading edge of cells.
The role of clathrin in pathogen entry has received much attention and has highlighted the adapta... more The role of clathrin in pathogen entry has received much attention and has highlighted the adaptability of clathrin during internalization. Recent studies have now uncovered additional roles for clathrin and have put the spotlight on its role in pathogen spread. Here, we discuss the manipulation of clathrin by pathogens, with specific attention to the processes that occur at the plasma membrane. In the majority of cases, both clathrin and the actin cytoskeleton are hijacked, so we also examine the interplay between these two systems and their role during pathogen internalization, egress and spread.
Vaccinia virus infection dramatically affects the host actin cytoskeleton by inducing disassembly... more Vaccinia virus infection dramatically affects the host actin cytoskeleton by inducing disassembly of actin stress fibres and formation of actin tails which propel the virus intra-and intercellularly. The viral factors responsible for these actin rearrangements remain unknown. Sequence analysis reveals significant homology between the vaccinia F8L ORF and the proline repeats of iActA, the protein which initiates actin tail assembly and motility in the bacterial pathogen Listeria ivanovii. We characterized the F8L gene product to examine its possible role in vaccinia rearrangements of the host actin cytoskeleton. F8L is a " 8 kDa protein expressed early during infection and is found throughout the cytoplasm, with no discernible association with viral or cellular structures. Furthermore, the F8L deletion strain, WR∆F8L, forms particles and actin tails indistinguishable from WR. Our observations demonstrate that F8L is not required for vaccinia virus morphogenesis or the actin rearrangements observed during infection.
The adaptor protein Nck has been shown to link receptor ligation to actin-based signalling in a d... more The adaptor protein Nck has been shown to link receptor ligation to actin-based signalling in a diverse range of cellular events, such as changes in cell morphology and motility. It has also been implicated in phagocytosis. However, its molecular role in controlling actin remodelling associated with phagocytic uptake remains to be clarified. Here, we show that Nck, which is recruited to phagocytic cups, is required for Fcc receptor (FccR)-but not complement receptor 3 (CR3)-induced phagocytosis. Nck recruitment in response to FccR ligation is mediated by the phosphorylation of tyrosine 282 and 298 in the ITAM motif in the cytoplasmic tail of the receptor. In the absence of FccR phosphorylation, there is also no recruitment of N-WASP or Cdc42 to phagocytic cups. Nck promotes FccR-mediated phagocytosis by recruiting N-WASP to phagocytic cups. Efficient phagocytosis, however, only occurs, if the CRIB domain of N-WASP can also interact with Cdc42. Our observations demonstrate that Nck and Cdc42 collaborate to stimulate N-WASP-dependent FccR-mediated phagocytosis.
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Papers by Michael Way