Re-evaluating the actin-dependence of spectraplakin functions during axon growth and maintenance

Axons are the long and slender processes of neurons constituting the biological cables that wire the nervous system. The growth and maintenance of axons require bundles of microtubules that extend through their entire length. Understanding microtubule regulation is therefore an essential aspect of axon biology. Key regulators of neuronal microtubules are the spectraplakins, a well-conserved family of cytoskeletal cross-linkers that underlie neuropathies in mouse and humans. Spectraplakin deficiency in mouse or Drosophila causes severe decay of microtubule bundles and axon growth inhibition. The underlying mechanisms are best understood for Drosophila Short stop (Shot) and believed to involve cytoskeletal cross-linkage: the N-terminal calponin homology (CH) domains bind to F-actin, and the C-terminus to microtubules and Eb1. Here we have gained new understanding by showing that the F-actin interaction must be finely balanced: altering the properties of F-actin networks or deleting/exchanging Shot's CH domains induces changes in Shot function - with a Lifeact-containing Shot variant causing remarkable remodelling of neuronal microtubules. In addition to actin-MT cross-linkage, we find strong indications that Shot executes redundant MT bundle-promoting roles that are F-actin-independent. We argue that these likely involve the neuronal Shot-PH isoform, which is characterised by a large, unexplored central plakin repeat region (PRR). Work on PRRs might therefore pave the way towards important new mechanisms of axon biology and architecture that might similarly apply to central PRRs in mammalian spectraplakins.

Drosophila Nesprin-1 Isoforms Differentially Contribute to Muscle Function

Nesprin-1 is a large scaffold protein connecting nuclei to the actin cytoskeleton via its KASH and Calponin Homology domains, respectively. Nesprin-1 disconnection from nuclei results in altered muscle function and myonuclei mispositioning. Furthermore, Nesprin-1 mutations are associated with muscular pathologies such as Emery Dreifuss muscular dystrophy and arthrogryposis. Nesprin-1 was thus proposed to mainly contribute to muscle function by controlling nuclei position. However, Nesprin-1′s localisation at sarcomere’s Z-discs, its involvement in organelles’ subcellular localization, as well as the description of numerous isoforms presenting different combinations of Calponin Homology (CH) and KASH domains, suggest that the contribution of Nesprin-1 to muscle functions is more complex. Here, we investigate the roles of Nesprin-1/Msp300 isoforms in muscle function and subcellular organisation using Drosophila larvae as a model. Subsets of Msp300 isoform were down-regulated by muscle-specific RNAi expression and muscle global function and morphology were assessed. We show that nuclei anchoring in mature muscle and global muscle function are disconnected functions associated with different Msp300 isoforms. Our work further uncovers a new and unsuspected role of Msp300 in myofibril registration and nuclei peripheral displacement supported by Msp300 CH containing isoforms, a function performed by Desmin in mammals.

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.

The Nesprin-1/-2 ortholog ANC-1 regulates organelle positioning in C. elegans independently from its KASH or actin-binding domains

KASH proteins in the outer nuclear membrane comprise the cytoplasmic half of LINC complexes that connect nuclei to the cytoskeleton. Caenorhabditis elegans ANC-1, an ortholog of Nesprin-1/2, contains actin-binding and KASH domains at opposite ends of a long spectrin-like region. Deletion of either the KASH or calponin homology (CH) domains does not completely disrupt nuclear positioning, suggesting neither KASH nor CH domains are essential. Deletions in the spectrin-like region of ANC-1 led to significant defects, but only recapitulated the null phenotype in combination with mutations in the trans-membrane span. In anc-1 mutants, the ER, mitochondria, and lipid droplets were unanchored, moving throughout the cytoplasm. The data presented here support a cytoplasmic integrity model where ANC-1 localizes to the ER membrane and extends into the cytoplasm to position nuclei, ER, mitochondria, and likely other organelles in place.

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.

Deciphering the Role of Filamin B Calponin-Homology Domain in Causing the Larsen Syndrome, Boomerang Dysplasia, and Atelosteogenesis Type I Spectrum Disorders via a Computational Approach

Filamins (FLN) are a family of actin-binding proteins involved in regulating the cytoskeleton and signaling phenomenon by developing a network with F-actin and FLN-binding partners. The FLN family comprises three conserved isoforms in mammals: FLNA, FLNB, and FLNC. FLNB is a multidomain monomer protein with domains containing an actin-binding N-terminal domain (ABD 1–242), encompassing two calponin-homology domains (assigned CH1 and CH2). Primary variants in FLNB mostly occur in the domain (CH2) and surrounding the hinge-1 region. The four autosomal dominant disorders that are associated with FLNB variants are Larsen syndrome, atelosteogenesis type I (AOI), atelosteogenesis type III (AOIII), and boomerang dysplasia (BD). Despite the intense clustering of FLNB variants contributing to the LS-AO-BD disorders, the genotype-phenotype correlation is still enigmatic. In silico prediction tools and molecular dynamics simulation (MDS) approaches have offered the potential for variant classification and pathogenicity predictions. We retrieved 285 FLNB missense variants from the UniProt, ClinVar, and HGMD databases in the current study. Of these, five and 39 variants were located in the CH1 and CH2 domains, respectively. These variants were subjected to various pathogenicity and stability prediction tools, evolutionary and conservation analyses, and biophysical and physicochemical properties analyses. Molecular dynamics simulation (MDS) was performed on the three candidate variants in the CH2 domain (W148R, F161C, and L171R) that were predicted to be the most pathogenic. The MDS analysis results showed that these three variants are highly compact compared to the native protein, suggesting that they could affect the protein on the structural and functional levels. The computational approach demonstrates the differences between the FLNB mutants and the wild type in a structural and functional context. Our findings expand our knowledge on the genotype-phenotype correlation in FLNB-related LS-AO-BD disorders on the molecular level, which may pave the way for optimizing drug therapy by integrating precision medicine.

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.

The Nesprin-1/-2 ortholog ANC-1 regulates organelle positioning in C. elegans without its KASH or actin-binding domains

KASH proteins in the outer nuclear membrane comprise the cytoplasmic half of LINC complexes that connect nuclei to the cytoskeleton. Caenorhabditis elegans ANC-1, an ortholog of Nesprin-1/2, contains actin-binding and KASH domains at opposite ends of a long spectrin-like region. Deletion of either the KASH or calponin homology (CH) domains does not completely disrupt nuclear positioning, suggesting neither KASH nor CH domains are essential. Deletions in the spectrin-like region of ANC-1 led to significant defects, but only recapitulated the null phenotype in combination with mutations in the trans-membrane span. In anc-1 mutants, the ER was unanchored, moving throughout the cytoplasm, and often fragmented. The data presented here support a cytoplasmic integrity model where ANC-1 localizes to the ER membrane and extends into the cytoplasm to position nuclei, ER, mitochondria, and likely other organelles in place.

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.