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1.  FHOD1 is needed for directed Forces and Adhesion Maturation during Cell Spreading and Migration 
Developmental cell  2013;27(5):545-559.
Summary
Matrix adhesions provide critical signals for cell growth or differentiation. They form through a number of distinct steps that follow integrin binding to matrix ligands. In an early step, integrins form clusters that support actin polymerization by an unknown mechanism. This raises the question of how actin polymerization occurs at the integrin clusters. We report here that a major formin in mouse fibroblasts, FHOD1 is recruited to integrin clusters, resulting in actin assembly. Using cell-spreading assays on lipid bilayers, solid substrates and high-resolution force sensing pillar arrays, we find that knockdown of FHOD1 impairs spreading, coordinated application of adhesive force and adhesion maturation. Finally we show that targeting of FHOD1 to the integrin sites depends on the direct interaction with Src family kinases, and is upstream of the activation by Rho Kinase. Thus our findings provide insights into the mechanisms of cell migration with implications for development and disease.
doi:10.1016/j.devcel.2013.11.003
PMCID: PMC3890431  PMID: 24331927
2.  Two distinct phosphorylation events govern the function of muscle FHOD3 
Posttranslational modifications such as phosphorylation are universally acknowledged regulators of protein function. Recently we characterised a striated muscle-specific isoform of the formin FHOD3 that displays distinct subcellular targeting and protein half-life compared to its non-muscle counterpart, which is dependent on phosphorylation by CK2 (formerly casein kinase 2). We now show that the two isoforms of FHOD3 are already expressed in the vertebrate embryonic heart. Analysis of CK2alpha knockout mice showed that phosphorylation by CK2 is required for proper targeting of muscle FHOD3 to the myofibrils also in embryonic cardiomyocytes in situ. The localisation of muscle FHOD3 in the sarcomere varies depending on the maturation state, being either broader or restricted to the Z-disc proper in adult heart. Following myofibril disassembly such as in dedifferentiating adult rat cardiomyocytes in culture, the expression of non-muscle FHOD3 is up-regulated, which is reversed once the myofibrils are reassembled. The shift in expression levels of different isoforms is accompanied by an increased co-localisation with p62, which is involved in autophagy, and affects the half-life of FHOD3.
Phosphorylation of three amino acids in the C-terminus of FHOD3 by ROCK1 is sufficient for activation, which results in increased actin filament synthesis in cardiomyocytes and also a broader localisation pattern of FHOD3 in the myofibrils. ROCK1 can directly phosphorylate FHOD3 and FHOD3 seems to be the downstream mediator of the exaggerated actin filament formation phenotype that is induced in cardiomyocytes upon the overexpression of constitutively active ROCK1. We conclude that the expression of the muscle FHOD3 isoform is characteristic for the healthy mature heart and that two distinct phosphorylation events are crucial to regulate its activity in thin filament assembly and maintenance.
doi:10.1007/s00018-012-1154-7
PMCID: PMC3696992  PMID: 23052206
myofibril; formin; cardiac cytoarchitecture; heart development
3.  Endoplasmic spreading requires coalescence of vimentin intermediate filaments at force-bearing adhesions 
Molecular Biology of the Cell  2013;24(1):21-30.
Interaction of vimentin filaments (vIFs) and force-bearing adhesions is essential for endoplasm spreading. For adhesions to be connected to a contractile network involved in endoplasm spreading, vIFs are needed. Thus endoplasm spreading and microtubule stabilization in the periphery require a multicomponent actin network anchored at adhesions.
For cells to develop long-range forces and carry materials to the periphery, the microtubule and organelle-rich region at the center of the cell—the endoplasm—needs to extend to near the cell edge. Depletion of the actin cross-linking protein filamin A (FlnA) causes a collapse of the endoplasm into a sphere around the nucleus of fibroblasts and disruption of matrix adhesions, indicating that FlnA is involved in endoplasmic spreading and adhesion growth. Here, we report that treatment with the calpain inhibitor N-[N-(N-acetyl-l-leucyl)-l-leucyl]-l-norleucine (ALLN) restores endoplasmic spreading as well as focal adhesion (FA) growth on fibronectin-coated surfaces in a Fln-depleted background. Addback of calpain-uncleavable talin, not full-length talin, achieves a similar effect in Fln-depleted cells and indicates a crucial role for talin in endoplasmic spreading. Because FA maturation involves the vimentin intermediate filament (vIF) network, we also examined the role of vIFs in endoplasmic spreading. Wild-type cells expressing a vimentin variant incapable of polymerization exhibit deficient endoplasmic spreading as well as defects in FA growth. ALLN treatment restores FA growth despite the lack of vIFs but does not restore endoplasmic spreading, implying that vIFs are essential for endoplasm spreading. Consistent with that hypothesis, vIFs are always displaced from adhesions when the endoplasm does not spread. In Fln-depleted cells, vIFs extend beyond adhesions, nearly to the cell edge. Finally, inhibiting myosin II–mediated contraction blocks endoplasmic spreading and adhesion growth. Thus we propose a model in which myosin II–mediated forces and coalescence of vIFs at mature FAs are required for endoplasmic spreading.
doi:10.1091/mbc.E12-05-0377
PMCID: PMC3530776  PMID: 23115305
4.  Formin follows function: a muscle-specific isoform of FHOD3 is regulated by CK2 phosphorylation and promotes myofibril maintenance 
The Journal of Cell Biology  2010;191(6):1159-1172.
Phosphorylation of the muscle-specific formin splice variant FHOD3 by CK2 regulates its stability, myofibril targeting, and myofibril integrity.
Members of the formin family are important for actin filament nucleation and elongation. We have identified a novel striated muscle–specific splice variant of the formin FHOD3 that introduces a casein kinase 2 (CK2) phosphorylation site. The specific targeting of muscle FHOD3 to the myofibrils in cardiomyocytes is abolished in phosphomutants or by the inhibition of CK2. Phosphorylation of muscle FHOD3 also prevents its interaction with p62/sequestosome 1 and its recruitment to autophagosomes. Furthermore, we show that muscle FHOD3 efficiently promotes the polymerization of actin filaments in cardiomyocytes and that the down-regulation of its expression severely affects myofibril integrity. In murine and human cardiomyopathy, we observe reduced FHOD3 expression with a concomitant isoform switch and change of subcellular targeting. Collectively, our data suggest that a muscle-specific isoform of FHOD3 is required for the maintenance of the contractile structures in heart muscle and that its function is regulated by posttranslational modification.
doi:10.1083/jcb.201005060
PMCID: PMC3002041  PMID: 21149568
5.  Formin-g muscle cytoarchitecture 
Bioarchitecture  2011;1(2):66-68.
Striated muscle cells display an extremely regular assembly of their actin cytoskeleton that contributes to the contractile elements, the myofibrils. How this assembly is initiated and how these structures are maintained is still unclear. We have recently shown that striated muscle expresses a specific isoform of the formin protein family member FHOD3, which is characterised by the presence of a CK2 phosphorylation site at the C-terminal end of the formin homology domain 2 (FH2). Phosphorylated muscle FHOD3 displays a different subcellular localisation, namely to the myofibrils, and also has increased stability compared to un-phosphorylated or non muscle FHOD3. In addition, we could show that muscle FHOD3 is involved in myofibril maintenance in cultured cardiomyocytes and that its presence dramatically enhances the reconstitution of cardiac actin filaments after depolymerisation. Since FHOD3 expression levels and in particular that of the muscle isoform are also decreased in different types of cardiomyopathy, we postulate a crucial role for this protein in the maintenance of a fully functional cardiac cytoarchitecture.
doi:10.4161/bioa.1.2.15467
PMCID: PMC3158626  PMID: 21866265
heart; development; actin filament; formin; sarcomere
6.  ABERRANT DEVELOPMENT OF NEUROMUSCULAR JUNCTIONS IN GLYCOSYLATION-DEFECTIVE LARGEMYD MICE 
Neuromuscular disorders : NMD  2009;19(5):366-378.
Mice deficient in the glycosyltransferase Large are characterized by severe muscle and central nervous system abnormalities. In this study, we show that the formation and maintenance of neuromuscular junctions in Largemyd mice are greatly compromised. Neuromuscular junctions are not confined to the muscle endplate zone but are widely spread and are frequently accompanied by exuberant nerve sprouting. Nerve terminals are highly fragmented and binding of α-bungarotoxin to postsynaptic acetylcholine receptors (AChRs) is greatly reduced. In vitro, Largemyd myotubes are responsive to agrin but produce aberrant AChR clusters, which are larger in area and less densely packed with AChRs. In addition, AChR expression on the cell surface is diminished suggesting that AChR assembly or transport is defective. These results together with the finding that O-linked glycosylation at neuromuscular junctions of Largemyd mice is compromised indicate that the action of Large is necessary for proper neuromuscular junction development.
doi:10.1016/j.nmd.2009.02.011
PMCID: PMC2958302  PMID: 19346129
glycosylation; neuromuscular junction; skeletal muscle; dystroglycan
7.  MAMMALIAN CELLS CONTAIN A SECOND NUCLEOCYTOPLASMIC HEXOSAMINIDASE 
The Biochemical journal  2009;419(1):83-90.
Some thirty years ago, work on mammalian tissues suggested the presence of two cytosolic hexosaminidases in mammalian cells; one of these has been more recently characterised in recombinant form and has an important role in cellular function due to its ability to cleave β-N-acetylglucosamine residues from a variety of nuclear and cytoplasmic proteins. However, the molecular nature of the second cytosolic hexosaminidase, named hexosaminidase D, has remained obscure. In the present study, we molecularly characterise for the first time the human and murine recombinant forms of enzymes, encoded by HEXDC genes, which appear to correspond to hexosaminidase D in terms of substrate specificity, pH dependency and temperature stability; furthermore, a myc-tagged form of this novel hexosaminidase displays a nucleocytoplasmic localisation. Transcripts of the corresponding gene are expressed in a number of murine tissues. Based on its sequence, this enzyme represents, along with the lysosomal hexosaminidase subunits encoded by the HEXA and HEXB genes, the third class 20 glycosidase to be found from mammalian sources.
doi:10.1042/BJ20081630
PMCID: PMC2850170  PMID: 19040401
N-acetylgalactosaminidase; nucleocytoplasmic; GFP; hexosaminidase; mouse; human

Results 1-7 (7)