Biomedical subjects

Nicholas H Brown

Publications and source records attributed to Nicholas H Brown.

At least 19 recordsLinked to original sources

PubMed2006

Integrins and the actin cytoskeleton.

The ability to connect to the actin cytoskeleton is a key part of the adhesive function of integrins. This linkage between integrins and the cytoskeleton involves a large complex of integrin-associated proteins that function in both the assembly and disassembly of the link. Genetic evidence has helped to clarify the relative contributions of different components of this link. In different contexts integrins can either stimulate or suppress actin based structures, indicating the variety of pathways leading from integrins to the cytoskeleton. The cytoskeleton also contributes to the extent of the integrin junction, allowing an adhesive contact to attain sufficient strength to resist contractile forces involved in cellular movement and function.

Actin Cytoskeleton↗

PubMed2006

The Drosophila RASSF homolog antagonizes the hippo pathway.

Correct organ size is determined by the balance between cell death and proliferation. Perturbation of this delicate balance leads to cancer formation . Hippo (Hpo), the Drosophila ortholog of MST1 and MST2 (Mammalian Sterile 20-like 1 and 2) is a key regulator of a signaling pathway that controls both cell death and proliferation . This pathway is so far composed of two Band 4.1 proteins, Expanded (Ex) and Merlin (Mer), two serine/threonine kinases, Hpo and Warts (Wts), the scaffold proteins Salvador (Sav) and Mats, and the transcriptional coactivator Yorkie (Yki). It has been proposed that Ex and Mer act upstream of Hpo, which in turn phosphorylates and activates Wts. Wts phosphorylates Yki and thus inhibits its activity and reduces expression of Yki target genes such as the caspase inhibitor DIAP1 and the micro RNA bantam. However, the mechanisms leading to Hpo activation are still poorly understood. In mammalian cells, members of the Ras association family (RASSF) of tumor suppressors have been shown to bind to MST1 and modulate its activity . In this study, we show that the Drosophila RASSF ortholog (dRASSF) restricts Hpo activity by competing with Sav for binding to Hpo. In addition, we observe that dRASSF also possesses a tumor-suppressor function.

Animals↗

PubMed2006

An interaction between integrin and the talin FERM domain mediates integrin activation but not linkage to the cytoskeleton.

Transmembrane adhesion receptors, such as integrins, mediate cell adhesion by interacting with intracellular proteins that connect to the cytoskeleton. Talin, one such linker protein, is thought to have two roles: mediating inside-out activation of integrins, and connecting extracellular matrix (ECM)-bound integrins to the cytoskeleton. Talin's amino-terminal head, which consists of a FERM domain, binds an NPxY motif within the cytoplasmic tail of most integrin beta subunits. This is consistent with the role of FERM domains in recruiting other proteins to the plasma membrane. We tested the role of the talin-head-NPxY interaction in integrin function in Drosophila. We found that introduction of a mutation that perturbs this binding in vitro into the isolated talin head disrupts its recruitment by integrins in vivo. Surprisingly, when engineered into the full-length talin, this mutation did not disrupt talin recruitment by integrins nor its ability to connect integrins to the cytoskeleton. However, it reduced the ability of talin to strengthen integrin adhesion to the ECM, indicating that the function of the talin-head-NPxY interaction is solely to regulate integrin adhesion.

Amino Acid Motifs↗

PubMed2006

Multiple factors contribute to integrin-talin interactions in vivo.

The cytoplasmic protein talin is an essential part of the integrin-cytoskeleton link. We characterized the interaction between integrin and two conserved regions of talin, the N-terminal ;head' domain and the C-terminus, which includes the I/LWEQ domain, within the living organism. Green-fluorescent-protein-tagged head and C-terminal domains were recruited to integrin adhesion sites. Both required integrins for recruitment, but the C-terminal domain also required endogenous talin, showing it was not recruited directly by integrins. We used chimeric transmembrane proteins containing the cytoplasmic domain of the integrin beta subunit to examine the integrin-talin head interaction. Monomeric chimeric proteins did not recruit talin head, whereas dimeric chimeras efficiently recruited it and caused a strong inhibition of integrin-mediated adhesion. These chimeras recruited surprisingly few integrin-associated proteins, indicating that recruitment of talin did not initiate a cascade of recruitment. Mutagenesis of the integrin cytoplasmic domain, within the chimera, showed the dominant-negative inhibition was not due to talin sequestration alone and that additional interactions are required.

Amino Acid Motifs↗

PubMed2005

Contribution of sequence variation in Drosophila actins to their incorporation into actin-based structures in vivo.

Actin is a highly conserved protein important for many cellular functions including motility, contraction in muscles and intracellular transport. Many eukaryotic genomes encode multiple actin protein isoforms that differ from each other by only a few residues. We addressed whether the sequence differences between actin paralogues in one species affect their ability to integrate into the large variety of structures generated by filamentous actin. We thus ectopically expressed all six Drosophila actins as fusion proteins with green fluorescent protein (GFP) in a variety of embryonic, larval and adult fly tissues. We found that each actin was able to integrate into most actin structures analysed. For example, in contrast to studies in mammalian cells, the two Drosophila cytoplasmic actins were incorporated into muscle sarcomeres. However, there were differences in the efficiency with which each actin was incorporated into specific actin structures. The most striking difference was observed within the Z-lines of the sarcomeres: one actin was specifically excluded and we mapped this feature to one or both of two residues within the C-terminal half of the protein. Thus, in Drosophila, the primary sequence of different actins does affect their ability to incorporate into actin structures, and so specific GFPactins may be used to label certain actin structures particularly well.

Actins↗

PubMed2005

Integrin-independent repression of cadherin transcription by talin during axis formation in Drosophila.

The Drosophila melanogaster anterior-posterior axis becomes polarized early during oogenesis by the posterior localization of the oocyte within the egg chamber. The invariant position of the oocyte is thought to be driven by an upregulation of the adhesion molecule DE-cadherin in the oocyte and the posterior somatic follicle cells, providing the first in vivo example of cell sorting that is specified by quantitative differences in cell-cell adhesion. However, it has remained unclear how DE-cadherin levels are regulated. Here, we show that talin, known for its role in linking integrins to the actin cytoskeleton, has the unexpected function of specifically inhibiting DE-cadherin transcription. Follicle cells that are mutant for talin show a strikingly high level of DE-cadherin, due to elevated transcription of DE-cadherin. We demonstrate that this deregulation of DE-cadherin is sufficient to attract the oocyte to lateral and anterior positions. Surprisingly, this function of talin is independent of integrins. These results uncover a new role for talin in regulating cadherin-mediated cell adhesion.

Actins↗

PubMed2005

Papillote and Piopio: Drosophila ZP-domain proteins required for cell adhesion to the apical extracellular matrix and microtubule organization.

Adhesion between epithelial cells and extracellular substrates is normally mediated through basal adhesion complexes. However, some cells also possess comparable junctions on their apical surface. Here, we describe two new Drosophila proteins, Piopio and Papillote, that are required for the link between the apical epithelial surface and the overlying apical extracellular matrix (aECM). The two proteins share a zona pellucida (ZP) domain with mammalian aECM components, including the tectorins found in the vertebrate inner ear. Tagged versions of both proteins localized to the apical epithelial surface. Mutations in piopio, papillote and dumpy (another gene encoding a ZP-domain protein) cause defects in the innermost layer of the aECM and its detachment from the epidermis. Loss of Piopio, but not Papillote or Dumpy, causes the absence of specialized microtubule bundles from pupal wings, suggesting that Piopio plays a role in microtubule organization. Thus, ZP domain-containing proteins may have shared functions within the aECM, while also exhibiting specific interactions with the cytoskeleton.

Amino Acid Sequence↗

PubMed2004

Focal adhesion kinase is not required for integrin function or viability in Drosophila.

The mammalian focal adhesion kinase (FAK) family of non-receptor protein-tyrosine kinases has been implicated in controlling a multitude of cellular responses to the engagement of cell-surface integrins and G-protein-coupled receptors. The high level of sequence conservation between the mammalian proteins and the Drosophila homologue of FAK, Fak56, suggested that it would have similar functions. However, we show here that Drosophila Fak56 is not essential for integrin functions in adhesion, migration or signaling in vivo. Furthermore, animals lacking Fak56 are viable and fertile, demonstrating that Fak56 is not essential for other developmental or physiological functions. Despite this, overexpressed Fak56 is a potent inhibitor of integrins binding to the extracellular matrix, suggesting that Fak56 may play a subtle role in the negative regulation of integrin adhesion.

Animals↗

PubMed2004

Morphogenesis in the absence of integrins: mutation of both Drosophila beta subunits prevents midgut migration.

Two integrin beta subunits are encoded in the Drosophila genome. The betaPS subunit is widely expressed and heterodimers containing this subunit are required for many developmental processes. The second betasubunit, betanu, is a divergent integrin expressed primarily in the midgut endoderm. To elucidate its function, we generated null mutations in the gene encoding betanu. We find that betanu is not required for viability or fertility, and overall the mutant flies are normal in appearance. However, we could observe betanu function in the absence of betaPS. Consistent with its expression, removal of betanu only enhanced the phenotype of betaPS in the developing midgut. In embryos lacking the zygotic contribution of betaPS, loss of betanu resulted in enhanced separation between the midgut and the surrounding visceral mesoderm. In the absence of both maternal and zygotic betaPS, a delay in midgut migration was observed, but removing betanu as well blocked migration completely. These results demonstrate that the second beta subunit can partially compensate for loss of betaPS integrins, and that integrins are essential for migration of the primordial midgut cells. The two beta subunits mediate midgut migration by distinct mechanisms: one that requires talin and one that does not. Other examples of developmental cell migration, such as that of the primordial germ cells, occurred normally in the absence of integrins. Having generated the tools to eliminate integrin function completely, we confirm that Drosophila integrins do not control proliferation as they do in mammals, and have identified alphaPS3 as a heterodimeric partner for betanu.

Animals↗

PubMed2004

Novel functions for integrins in epithelial morphogenesis.

Dorsal closure during Drosophila embryogenesis provides a valuable model for epithelial morphogenesis and wound healing. Previous studies have focused on two cell populations, the dorsal epidermis and the extraembryonic amnioserosa. Here, we demonstrate that there is an additional player, the large yolk cell. We find that integrins are expressed in the amnioserosa and yolk cell membrane and that they are required for three processes: (1) assembly of an intervening extracellular matrix, (2) attachment between these two cell layers, and (3) contraction of the amnioserosa cells. We also provide evidence for integrin-extracellular matrix interactions occurring between the lateral surfaces of the amnioserosa cell and the leading edge epidermis that effectively mediate cell-cell adhesion. Thus, dorsal closure shares mechanistic similarities with vertebrate epithelial morphogenetic events, including epiboly, that also employ an underlying substrate.

Animals↗

PubMed2004

A spectraplakin is enriched on the fusome and organizes microtubules during oocyte specification in Drosophila.

BACKGROUND: During Drosophila oogenesis a membranous organelle called the fusome has a key function in the establishment of oocyte fate and polarity, ultimately leading to the establishment of the major body axes of the animal. The fusome is necessary for the microtubule-driven restriction of markers of oocyte fate to the oocyte, but the mechanism by which the fusome organizes the microtubules is not known. RESULTS: We have identified the spectraplakin Short stop (Shot) as a new component of the fusome. Spectraplakins are giant cytoskeletal linker proteins, with multiple isoforms produced from each gene. Shot is the sole spectraplakin in Drosophila. The phenotype caused by the absence of Shot is not similar to that of other components of the fusome but instead is similar to the absence of the downstream components that interact with microtubules: the dynein/dynactin-complex-associated proteins Egalitarian and BicaudalD. Shot is required for the association of microtubules with the fusome and the subsequent specification of the oocyte in 16-cell cysts. Shot is also required for the concentration of centrosomes into the oocyte, a process thought to be independent of microtubules because it still occurs in the presence of microtubule depolymerizing drugs. This suggests that Shot may protect some microtubules from depolymerization and that these microtubules are sufficient for this process. CONCLUSIONS: Shot provides the missing link between the fusome and microtubules within meiotic cysts, which is essential for the establishment of the oocyte. Shot associates with the fusome and is required for microtubule organization. We suggest that it does this directly, via its microtubule binding GAS2 domain.

Animals↗

PubMed2004

Tensin stabilizes integrin adhesive contacts in Drosophila.

We report the functional characterization of the Drosophila ortholog of tensin, a protein implicated in linking integrins to the cytoskeleton and signaling pathways. A tensin null was generated and is viable with wing blisters, a phenotype characteristic of loss of integrin adhesion. In tensin mutants, mechanical abrasion is required during wing expansion to cause wing blisters, suggesting that tensin strengthens integrin adhesion. The localization of tensin requires integrins, talin, and integrin-linked kinase. The N-terminal domain and C-terminal PTB domain of tensin provide essential recruitment signals. The intervening SH2 domain is not localized on its own. We suggest a model where tensin is recruited to sites of integrin adhesion via its PTB and N-terminal domains, localizing the SH2 domain so that it can interact with phosphotyrosine-containing proteins, which stabilize the integrin link to the cytoskeleton.

Animals↗

PubMed2003

Reducing integrins improves the quality of fly life.

Integrins are cell surface receptors that mediate adhesion to the extracellular matrix. In this Perspective, we focus on the recent finding that a decrease in the levels of integrins yields fruit flies that retain youthful mobility as they age and display an increased mean life span. We discuss how the reduction of integrin activity might lead to the observed changes.

Animals↗

PubMed2003

Maintaining epithelial integrity: a function for gigantic spectraplakin isoforms in adherens junctions.

The Short stop (Shot/Kakapo) spectraplakin is a giant cytoskeletal protein, which exists in multiple isoforms with characteristics of both spectrin and plakin superfamilies. Previously characterized Shot isoforms are similar to spectrin and dystrophin, with an actin-binding domain followed by spectrin repeats. We describe a new large exon within the shot locus, which encodes a series of plakin repeats similar to the COOH terminus of plakins such as plectin and BPAG1e. We find that the plakin repeats are inserted between the actin-binding domain and spectrin repeats, generating isoforms as large as 8,846 residues, which could span 400 nm. These novel isoforms localized to adherens junctions of embryonic and follicular epithelia. Loss of Shot within the follicle epithelium leads to double layering and accumulation of actin and ZO-1 in between, and a reduction of Armadillo and Discs lost within, mutant cells, indicative of a disruption of adherens junction integrity. Thus, we identify a new role for spectraplakins in mediating cell-cell adhesion.

Adherens Junctions↗

PubMed2002

The 'spectraplakins': cytoskeletal giants with characteristics of both spectrin and plakin families.

Recent studies have characterised a family of giant cytoskeletal crosslinkers encoded by the short stop gene in Drosophila and the dystonin/BPAG1 and MACF1 genes in mammals. We refer to the products of these genes as spectraplakins to highlight the fact that they share features with both the spectrin and plakin superfamilies. These genes produce a variety of large proteins, up to almost 9000 residues long, which can potentially extend 0.4 micro m across a cell. Spectraplakins can interact with all three elements of the cytoskeleton: actin, microtubules and intermediate filaments. The analysis of mutant phenotypes in BPAG1 in mouse and short stop in Drosophila demonstrates that spectraplakins have diverse roles. These include linking the plasma membrane and the cytoskeleton, linking together different elements of the cytoskeleton and organising membrane domains.

Actins↗

PubMed2002

Integrin adhesion: when is a kinase a kinase?

Recent genetic studies in the worm Caenorhabditis elegans and fruitfly Drosophila have revealed the essential role integrin-linked kinase plays in integrin adhesion - but it apparently acts in this role as an adaptor rather than a kinase.

Animals↗

PubMed2002

Rap1 GTPase regulation of adherens junction positioning and cell adhesion.

Cell-cell junctions are distributed evenly around the lateral circumference of cells within an epithelium. We find that the even distribution of adherens junctions is an active process that requires the small guanosine triphosphatase Rap1. Cells mutant for Rap1 condensed their adherens junctions to one side of the cell. This disrupted normal epithelial cell behavior, and mutant cell clones dispersed into the surrounding wild-type tissue. Rap1 is enriched at adherens junctions, particularly between newly divided sister cells where it may reseal the adherens junction ring. The regulation of adherens junction positioning could play a role in cell mobility and cell division.

Adherens Junctions↗

PubMed2002

Integrins in development: moving on, responding to, and sticking to the extracellular matrix.

Integrins are cell surface receptors of the extracellular matrix present in all animals. Genetic analysis in worms, flies, and vertebrates has revealed integrin involvement in key developmental processes, and we focus here on examples of integrin functions that are comparable across these model organisms. Integrins contribute to cell movement by providing traction to migrating cells, through assembly of extracellular matrices that can serve as tracks for migration, and by transmitting guidance signals that direct cells or cell processes to their targets. Integrins also participate in signaling events that govern tissue differentiation and organogenesis. Finally, adhesion by integrin-mediated junctions allows tissues to withstand mechanical load and is essential for tissue integrity.

Animals↗