Calponin-homology domain mediated bending of membrane associated actin filaments

Actin filaments are central to numerous biological processes in all domains of life. Driven by the interplay with molecular motors, actin binding and actin modulating proteins, the actin cytoskeleton exhibits a variety of geometries. This includes structures with a curved geometry such as axon-stabilizing actin rings, actin cages around mitochondria and the cytokinetic actomyosin ring, which are generally assumed to be formed by short linear filaments held together by actin cross-linkers. However, whether individual actin filaments in these structures could be curved and how they may assume a curved geometry remains unknown. Here, we show that 'curly', a region from the IQGAP family of proteins from three different organisms, comprising the actin-binding calponin-homology domain and a C-terminal unstructured domain, stabilizes individual actin filaments in a curved geometry when anchored to lipid membranes. Whereas F-actin is semi-flexible with a persistence length of ~10 mm, binding of mobile curly within lipid membranes generates actin filament arcs and full rings of high curvature with radii below 1 mm. Higher rates of fully formed actin rings are observed in the presence of the actin-binding coiled-coil protein tropomyosin and when actin is directly polymerized on lipid membranes decorated with curly. Strikingly, curly induced actin filament rings contract upon the addition of muscle myosin II filaments and expression of curly in mammalian cells leads to highly curved actin structures in the cytoskeleton. Taken together, our work identifies a new mechanism to generate highly curved actin filaments, which opens a range of possibilities to control actin filament geometries, that can be used, for example, in designing synthetic cytoskeletal structures.

A Tug-of-War Model Explains the Saltatory Sperm Cell Movement in Arabidopsis thaliana Pollen Tubes by Kinesins With Calponin Homology Domain

Upon pollination, two sperm cells are transported inside the growing pollen tube toward the apex. One sperm cell fertilizes the egg cell to form the zygote, while the other fuses with the two polar nuclei to form the triploid endosperm. In Arabidopsis thaliana, the transport of the two sperm cells is characterized by sequential forward and backward movements with intermediate pauses. Until now, it is under debate which components of the plant cytoskeleton govern this mechanism. The sperm cells are interconnected and linked to the vegetative nucleus via a cytoplasmic projection, thus forming the male germ unit. This led to the common hypothesis that the vegetative nucleus is actively transported via myosin motors along actin cables while pulling along the sperm cells as passive cargo. In this study, however, we show that upon occasional germ unit disassembly, the sperm cells are transported independently and still follow the same bidirectional movement pattern. Moreover, we found that the net movement of sperm cells results from a combination of both longer and faster runs toward the pollen tube apex. We propose that the observed saltatory movement can be explained by the function of kinesins with calponin homology domain (KCH). This subgroup of the kinesin-14 family actively links actin filaments and microtubules. Based on KCH's specific properties derived from in vitro experiments, we built a tug-of-war model that could reproduce the characteristic sperm cell movement in pollen tubes.

EH domain binding protein 1-like 1 (EHBP1L1), a protein with calponin homology domain, is expressed in the rat testis

SummaryIn this paper, with the aim to find new genes involved in mammalian spermatogenesis, we isolated, for the first time in the rat testis, a partial cDNA clone that encoded EH domain binding protein 1-like 1 (Ehbp1l1), a protein that has a single calponin homology domain (CH). Bioinformatic analysis showed that EHBP1l1 contains three domains: the N-terminal C2-like, the CH and the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domains, which are evolutionarily conserved in vertebrates. We found that Ehbp1l1 mRNA was expressed in several rat tissues, including the liver, intestine, kidney and also in the testis during its development, with a higher level in testis from 12-month-old animals. Interestingly, in situ hybridization experiments revealed that Ehbp1l1 is specifically expressed by types I and II spermatocytes, this result was validated by RT-PCR performed on total RNA obtained from enriched fractions of different testicular cell types. As EHBP1l1 has been described as linked to vesicular transport to the actin cytoskeleton and as an effector of the small GTPase Rab8, we hypothesized that it could participate both in cytoskeletal remodelling and in the regulation of vesicle sorting from the trans-Golgi network to the apical plasma membrane. Our findings provide a better understand of the molecular mechanisms of the differentiation process of spermatogenesis; Ehbp1l1 may also be used as a new marker of testicular activity.

Calponin-Homology Domain mediated bending of membrane associated actin filaments

Actin filaments are central to numerous biological processes in all domains of life. Driven by the interplay with molecular motors, actin binding and actin modulating proteins, the actin cytoskeleton exhibits a variety of geometries. This includes structures with a curved geometry such as axon-stabilizing actin rings, actin cages around mitochondria and the cytokinetic actomyosin ring, which are generally assumed to be formed by short linear filaments held together by actin cross-linkers. However, whether individual actin filaments in these structures could be curved and how they may assume a curved geometry remains unknown. Here, we show that “curly”, a region from the IQGAP family of proteins from three different organisms, comprising the actin-binding calponin-homology domain and a C-terminal unstructured domain, stabilizes individual actin filaments in a curved geometry when anchored to lipid membranes. Whereas F-actin is semi-flexible with a persistence length of ∼10 μm, binding of mobile curly within lipid membranes generates actin filament arcs and full rings of high curvature with radii below 1 μm. Higher rates of fully formed actin rings are observed in the presence of the actin-binding coiled-coil protein tropomyosin, and also when actin is directly polymerized on lipid membranes decorated with curly. Strikingly, curly induced actin filament rings contract upon the addition of muscle myosin II filaments and expression of curly in mammalian cells leads to highly curved actin structures in the cytoskeleton. Taken together, our work identifies a new mechanism to generate highly curved actin filaments, which opens a new range of possibilities to control actin filament geometries, that can be used, for example, in designing synthetic cytoskeletal structures.

Steric regulation of tandem calponin homology domain actin-binding affinity

We show that the affinity of CH1–CH2 domains for F-actin can be both increased and decreased by diverse modifications that change the effective “openness” of CH1 and CH2, which sterically regulates binding to F-actin. We also show that subcellular localization depends on the N-terminal flanking region of CH1 but not on the overall affinity for F-actin.

Endothelial IQGAP1 regulates leukocyte transmigration by directing the LBRC to the site of diapedesis

Transendothelial migration (TEM) of leukocytes across the endothelium is critical for inflammation. In the endothelium, TEM requires the coordination of membrane movements and cytoskeletal interactions, including, prominently, recruitment of the lateral border recycling compartment (LBRC). The scaffold protein IQGAP1 was recently identified in a screen for LBRC-interacting proteins. Knockdown of endothelial IQGAP1 disrupted the directed movement of the LBRC and substantially reduced leukocyte TEM. Expression of truncated IQGAP1 constructs demonstrated that the calponin homology domain is required for IQGAP1 localization to endothelial borders and that the IQ domain, on the same IQGAP1 polypeptide, is required for its function in TEM. This is the first reported function of IQGAP1 requiring two domains to be present on the same polypeptide. Additionally, we show for the first time that IQGAP1 in the endothelium is required for efficient TEM in vivo. These findings reveal a novel function for IQGAP1 and demonstrate that IQGAP1 in endothelial cells facilitates TEM by directing the LBRC to the site of TEM.