FHOD1 regulates stress fiber organization by controlling transversal arc and dorsal fiber dynamics

Abstract

The formin FHOD1 (formin homology 2 domain containing protein 1) can act as a capping and bundling protein in vitro. In cells, active FHOD1 stimulates the formation of ventral stress fibers. However, the cellular mechanisms by which this phenotype is produced and the physiological relevance of FHOD1 function are not currently understood. Here, we first show that FHOD1 controls the formation of two distinct stress fiber precursors differentially. On the one hand, it inhibits dorsal fiber growth, which requires the polymerization of parallel bundles of long actin filaments. On the other hand, it stimulates transverse arcs that are formed by the fusion of short antiparallel actin filaments. This combined action is crucial for the maturation of stress fibers and their spatio-temporal organization, and a lack of FHOD1 function perturbs dynamic cell behavior during cell migration. Furthermore, we show that the GTPase-binding and formin homology 3 domains (GBD and FH3) are responsible for stress fiber association and colocalization with myosin. Surprisingly, a version of FHOD1 that lacks these domains nevertheless retains its full capacity to stimulate arc and ventral stress fiber formation. Based on our findings, we propose a mechanism in which FHOD1 promotes the formation of short actin filaments and transiently associates with transverse arcs, thus providing tight temporal and spatial control of the formation and turnover of transverse arcs into mature ventral stress fibers during dynamic cell behavior.

The formins FHOD1 and INF2 regulate inter- and intra-structural contractility of podosomes

“…In a reciprocal experiment, expression of a non-autoinhibited form of FHOD1, lacking the C-terminal DAD domain, led to pronounced formation of connecting cables, their prominent decoration with myosin IIA, and to increased levels of p-MLC at podosomes. These results are also in line with current literature showing an impact of FHOD1 in the formation of actin cables in several cell types, as expression of a dominant active form of the Drosophila homolog Knittrig led to stress fiber formation in macrophages and endothelial cells (Lammel et al, 2014), and expression of non-autoinhibited FHOD1-V228E led to increased formation of stress fibers in U2OS osteosarcoma cells (Schulze et al, 2014).…”

“…Control cells showed increasing pMLC levels during the reformation phase, which probably reflects the not-yet balanced forces between podosomes during the establishment of the equidistant podosome pattern. These results fit well with the observed preference of both myosin IIA (Verkhovsky and Borisy, 1993) and FHOD1 (Schulze et al, 2014) to bind to anti-parallel, i.e. potentially contractile, actin filaments.…”

“…In addition, the ability of FHOD1 to inhibit the growth of parallel, i.e. non-contractile actin filaments (Schulze et al, 2014), might help to ensure that only anti-parallel bundles of actin are formed between podosomes. Depletion of FHOD1 might thus also lead to enhanced formation of parallel actin filaments between podosomes.…”

“…FHOD1 has emerged as a regulator of stress fibers (Koka et al, 2003), through regulating the dynamics of actin transverse arcs and dorsal fibers (Schulze et al, 2014). Notably, FHOD1 has recently been shown to regulate maturation of integrin-based adhesion sites (Iskratsch et al, 2013).…”

Tropomyosin – master regulator of actin filament function in the cytoskeleton

“…Tpm1.6 decorates the entire length of dorsal stress fibers, whereas, Tpm2.1, Tpm3.1 and Tpm3.2 localize only to the dorsal stress fiber segment that overlaps with focal adhesions (Tojkander et al, 2011). It is also interesting to note, that several formin proteins, including Dia1, Dia2, Daam1 and FHOD1, are linked to stress fiber assembly (Hotulainen and Lappalainen, 2006;Ang et al, 2010;Tojkander et al, 2011;Schulze et al, 2014). Thus, it is possible that distinct formin-Tpm pairs specify different actin filament populations within the stress fiber network, similarly to what has recently been demonstrated in fission yeast for other types of actin filament structure (Johnson et al, 2014) (Fig.…”