Calponin Homology Research Articles

The Shot CH1 domain recognises a distinct form of F-actin during Drosophila oocyte determination.

In Drosophila, only one cell in a multicellular female germline cyst is specified as an oocyte and a similar process occurs in mammals. The symmetry-breaking cue for oocyte selection is provided by the fusome, a tubular structure connecting all cells in the cyst. The Drosophila spectraplakin Shot localises to the fusome and translates its asymmetry into a polarised microtubule network that is essential for oocyte specification, but how Shot recognises the fusome is unclear. Here, we demonstrate that the actin-binding domain (ABD) of Shot is necessary and sufficient to localise Shot to the fusome and mediates Shot function in oocyte specification together with the microtubule-binding domains. The calponin homology domain 1 of the Shot ABD recognises fusomal F-actin and requires calponin homology domain 2 to distinguish it from other forms of F-actin in the cyst. By contrast, the ABDs of utrophin, Fimbrin, Filamin, Lifeact and F-tractin do not recognise fusomal F-actin. We therefore propose that Shot propagates fusome asymmetry by recognising a specific conformational state of F-actin on the fusome.

1H, 13C, and 15N resonance assignments and solution structure of the N-terminal divergent calponin homology (NN-CH) domain of human intraflagellar transport protein 54.

The intraflagellar transport (IFT) machinery plays a crucial role in the bidirectional trafficking of components necessary for ciliary signaling, such as the Hedgehog, Wnt/PCR, and cAMP/PKA systems. Defects in some components of the IFT machinery cause dysfunction, leading to a wide range of human diseases and developmental disorders termed ciliopathies, such as nephronophthisis. The IFT machinery comprises three sub-complexes: BBsome, IFT-A, and IFT-B. The IFT protein 54 (IFT54) is an important component of the IFT-B sub-complex. In anterograde movement, IFT54 binds to active kinesin-II, walking along the cilia microtubule axoneme and carrying the dynein-2 complex in an inactive state, which works for retrograde movement. Several mutations in IFT54 are known to cause Senior-Loken syndrome, a ciliopathy. IFT54 possesses a divergent Calponin Homology (CH) domain termed as NN-CH domain at its N-terminus. However, several aspects of the function of the NN-CH domain of IFT54 are still obscure. Here, we report the 1H, 15N, and 13C resonance assignments of the NN-CH domain of human IFT54 and its solution structure. The NN-CH domain of human IFT54 adopts essentially the α1-α2-α3-α4-α5 topology as that of mouse IFT54, whose structure was determined by X-ray crystallographic study. The structural information and assignments obtained in this study shed light on the molecular function of the NN-CH domain in IFT54.

Interference of the Circular RNA Sperm Antigen With Calponin Homology and Coiled-Coil Domains 1 Suppresses Growth and Promotes Apoptosis of Breast Cancer Cells Partially Through Targeting miR-1236-3p/Chromobox 8 Pathway

Noncoding RNAs and RNA modifiers are implicated in cancer radiotherapy. Here, we aimed to investigate the role of sperm antigen with calponin homology and coiled-coil domains 1 (SPECC1)-derived circular RNA (circSPECC1; hsa_circ_0000745) in breast cancer (BC) cells under radiation treatment. Based on quantitative real-time PCR, circSPECC1 was highly upregulated in BC patients’ tumors and cells, and circSPECC1 expression was further increased with the dosage of radiation in BC cells. Moreover, circSPECC1 upregulation was found to be concomitant with higher chromobox 8 (CBX8) and lower microRNA (miR)-1236-3p expression. Functionally, 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT), 5-ethynyl-2′-deoxyuridine (EdU) and colony formation assays showed that circSPECC1 interference suppressed cell proliferation and long-term survival in BC cells and irradiated BC cells. Xenograft tumor model experiments showed that circSPECC1 knockdown restrained BC tumor growth in vivo. Meanwhile, flow cytometry assay and western blotting revealed an enhanced apoptosis by silencing circSPECC1. Moreover, miR-1236-3p overexpression, similar to circSPECC1 silencing, displayed anti-growth and proapoptosis roles in irradiated BC cells. Mechanistically, dual-luciferase reporter assay and RNA immunoprecipitation assay identified a target relationship between miR-1236-3p and circSPECC1 or CBX8. Also, CBX8 expression could be modulated by circSPECC1 via miR-1236-3p regulation. Collectively, we indicated that inhibiting circSPECC1 could suppress growth and promote apoptosis of BC cells in both irradiated and nonirradiated conditions at least partially via miR-1236-3p/CBX8 axis, confirming that circSPECC1 might be target to develop anticancer drug in BC.

Unraveling the mechanisms and evolution of a two-domain module in IQGAP proteins for controlling eukaryotic cytokinesis

The IQGAP family of proteins plays a crucial role in cytokinesis across diverse organisms, but the underlying mechanisms are not fully understood. In this study, we demonstrate that IQGAPs in budding yeast, fission yeast, and human cells use a two-domain module to regulate their localization as well as the assembly and disassembly of the actomyosin ring during cytokinesis. Strikingly, the calponin homology domains (CHDs) in these IQGAPs bind to distinct cellular F-actin structures with varying specificity, whereas the non-conserved domains immediately downstream of the CHDs in these IQGAPs all target the division site, but differ in timing, localization strength, and binding partners. We also demonstrate that human IQGAP3 acts in parallel to septins and myosin-IIs to mediate the role of anillin in cytokinesis. Collectively, our findings highlight the two-domain mechanism by which IQGAPs regulate cytokinesis in distantly related organisms as well as their evolutionary conservation and divergence.

Bioinformatic analysis of smoothelin family members supports tissue-specific functions of unique C-terminal calponin homology domains.

Smoothelins are cytoskeletal proteins with a single C-terminal calponin homology domain type 2 (CHD2). Little is known about the significance of variation in SMTN CHD2 domains, addressed here through analysis of public databases. A conserved 152 nt penultimate constitutive exon present in all SMTNs encodes helices II-IV of CHD2 with high identity (nt/aa 63/65%). Variable CHD2s of SMTN (helices IV-VI) are generated by alternative splicing of 165 nt exon E20. E20 and the CHD2 it encodes have high homology with the terminal constitutive exon of SMTNL1 (E8; nt/aa 72/75% identity). Unique to these CHD2 variants are a conserved extended nine amino acid C-terminal tail containing KTKK ubiquitination motifs. When E20 of SMTN is skipped (SMTN E20-), constitutive terminal E21 codes for helices IV-VI of CHD2. SMTN E21 has high identity with the terminal exon of SMTNL2 (E8; nt/aa 75/81% identity of aligned sequences) except for coding for a unique extended C-terminus (24 nt; 8aa) conserved only in mammals. SMTN isoform expression is tissue-specific: SMTNE20- and SMTNE20+ are highly expressed in SMC and non-muscle cells, respectively, while SMTNL1 + 2 are highly expressed in skeletal muscle cells. Tissue-specific expression of SMTN CHD2s with unique helices IV-VI suggest tissue-specific functions that require further study.

Calponin homology domain containing kinesin, KIS1, regulates chloroplast stromule formation and immunity.

Chloroplast morphology changes during immunity, giving rise to tubule-like structures known as stromules. Stromules extend along microtubules and anchor to actin filaments along nuclei to promote perinuclear chloroplast clustering. This facilitates the transport of defense molecules/proteins from chloroplasts to the nucleus. Evidence for a direct role for stromules in immunity is lacking since, currently, there are no known genes that regulate stromule biogenesis. We show that a calponin homology (CH) domain containing kinesin, KIS1 (kinesin required for inducing stromules 1), is required for stromule formation during TNL [TIR (Toll/Interleukin-1 receptor)-type nucleotide-binding leucine-rich repeat]-immune receptor-mediated immunity. Furthermore, KIS1 is required for TNL-mediated immunity to bacterial and viral pathogens. The microtubule-binding motor domain of KIS1 is required for stromule formation while the actin-binding, CH domain is required for perinuclear chloroplast clustering. We show that KIS1 functions through early immune signaling components, EDS1 and PAD4, with salicylic acid-induced stromules requiring KIS1. Thus, KIS1 represents a player in stromule biogenesis.

Specc1l deficiency leads to abnormal oocyte meiosis and reduced blastocyst development in mouse.

This study aimed to investigate the potential role of the mouse Specc1l (Sperm antigen with calponin homology and coiled-coil domains 1 like), a microtubule and actin cytoskeleton-associated protein during oocyte meiotic maturation and its potential effects on preimplantation development. This study shows that the transcriptional levels of Specc1l did not significantly change from the germinal vesicle (GV) stage to the metaphase II (MII) stage, but maternal transcripts rapidly and gradually degraded after fertilization. Specc1l was detected in both the cytoplasm and GV, but not in the nucleolus-like body in the GV intact oocyte. At the MII stages, Specc1l was widely distributed in the cytoplasm but did not co-localize with chromatin. Knockdown of Specc1l expression in oocytes resulted in abnormal spindle morphology and misaligned chromosomes, as well as a decrease in the rate of polar body extrusion and a reduced developmental competence of oocytes, leading to decreased blastocyst formation rate. In conclusion, this study provides evidence that Specc1l plays a critical role in mouse oocyte meiotic maturation and early embryo development, specifically in proper bipolar spindle assembly and extrusion of the first polar body.

Increased Actin Binding Is a Shared Molecular Consequence of Numerous SCA5 Mutations in β-III-Spectrin.

Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the SPTBN2 gene encoding the cytoskeletal protein β-III-spectrin. Previously, we demonstrated that a L253P missense mutation, localizing to the β-III-spectrin actin-binding domain (ABD), causes increased actin-binding affinity. Here we investigate the molecular consequences of nine additional ABD-localized, SCA5 missense mutations: V58M, K61E, T62I, K65E, F160C, D255G, T271I, Y272H, and H278R. We show that all of the mutations, similar to L253P, are positioned at or near the interface of the two calponin homology subdomains (CH1 and CH2) comprising the ABD. Using biochemical and biophysical approaches, we demonstrate that the mutant ABD proteins can attain a well-folded state. However, thermal denaturation studies show that all nine mutations are destabilizing, suggesting a structural disruption at the CH1-CH2 interface. Importantly, all nine mutations cause increased actin binding. The mutant actin-binding affinities vary greatly, and none of the nine mutations increase actin-binding affinity as much as L253P. ABD mutations causing high-affinity actin binding, with the notable exception of L253P, appear to be associated with an early age of symptom onset. Altogether, the data indicate that increased actin-binding affinity is a shared molecular consequence of numerous SCA5 mutations, which has important therapeutic implications.

End-binding protein 1 promotes specific motor-cargo association in the cell body prior to axonal delivery of dense core vesicles

Axonal transport is key to neuronal function. Efficient transport requires specific motor-cargo association in the soma, yet the mechanisms regulating this early step remain poorly understood. We found that EBP-1, the C.elegans ortholog of the canonical-microtubule-end-binding protein EB1, promotes the specific association between kinesin-3/KIF1A/UNC-104 and dense core vesicles (DCVs) prior to their axonal delivery. Using single-neuron, invivo labeling of endogenous cargo and EBs, we observed reduced axonal abundance and reduced secretion of DCV cargo, but not other KIF1A/UNC-104 cargoes, in ebp-1 mutants. This reduction could be traced back to fewer exit events from the cell body, where EBP-1 colocalized with the DCV sorting machinery at the trans Golgi, suggesting that this is the site of EBP-1 function. EBP-1 calponin homology (CH) domain was required for directing microtubule growth on the Golgi, and mammalian EB1 interacted with KIF1A in an EBH-domain-dependent manner. Loss- and gain-of-function experiments suggest a model in which both kinesin-3 binding and guidance of microtubule growth at the trans Golgi by EBP-1 promote motor-cargo association at sites of DCV biogenesis. In support of this model, tethering either EBP-1 or a kinesin-3/KIF1A/UNC-104-interacting domain from an unrelated protein to the Golgi restored the axonal abundance of DCV proteins in ebp-1 mutants. These results uncover an unexpected role for a microtubule-associated protein and provide insights into how specific kinesin-3 cargo is delivered to the axon.

SPECC1L: a cytoskeletal protein that regulates embryonic tissue dynamics.

Many structural birth defects occur due to failure of tissue movement and fusion events during embryogenesis. Examples of such birth defects include failure of closure of the neural tube, palate, and ventral body wall. Actomyosin forces play a pivotal role in these closure processes, making proteins that regulate actomyosin dynamics a priority when studying the etiology of structural birth defects. SPECC1L (sperm antigen with calponin homology and coiled-coil domains 1 like) cytoskeletal protein associates with microtubules, filamentous actin, non-muscle myosin II (NMII), as well as membrane-associated components of adherens junctions. Patients with SPECC1L mutations show a range of structural birth defects affecting craniofacial development (hypertelorism, cleft palate), ventral body wall (omphalocele), and internal organs (diaphragmatic hernia, bicornuate uterus). Characterization of mouse models indicates that these syndromic mutations utilize a gain-of-function mechanism to affect intra- and supra-cellular actin organization. Interestingly, SPECC1L deficiency appears to affect the efficiency of tissue dynamics, making it an important cytoskeletal regulator to study tissue movement and fusion events during embryonic development. Here we summarize the SPECC1L-related syndrome mutations, phenotypes of Specc1l mouse models, and cellular functions of SPECC1L that highlight how it may regulate embryonic tissue dynamics.

Circular RNA circLRCH3 promotes oxaliplatin resistance in gastric cancer through the modulation of the miR-383-5p/FGF7 axis.

Gastric cancer (GC) is a common malignant tumor of the digestive system. Circular RNAs (circRNAs) play a vital role in tumorigenesis and chemoresistance. The current study aimed to explore the possible role and mechanism of circRNA leucine rich repeats and calponin homology domain containing 3 (circLRCH3) in GC chemoresistance. The levels of circLRCH3, microRNA-383-5p (miR-383-5p) and fibroblast growth factor 7 (FGF7) were determined by quantitative real-time PCR or Western blot. Cell Counting Kit-8 (CCK-8) assay was utilized to evaluate cell survival rate and proliferation ability. Colony formation, transwell and flow cytometry assays were used to assess cell proliferation, migration, invasion and apoptosis. The expression of multidrug resistance proteins was detected by Western blot. The binding relationship between miR-383-5p and circLRCH3/FGF7 was verified by dual-luciferase reporter assay or RNA immunoprecipitation assay. Xenograft assay was conducted to analyze the role of circLRCH3 in OXA resistance in vivo. CircLRCH3 and FGF7 levels were up-regulated, while miR-383-5p level was reduced in OXA-resistant GC tissues and cells. Depletion of circLRCH3 attenuated the resistance of OXA-resistant cells to OXA. CircLRCH3 silence reduced OXA resistance by regulating miR-383-5p. Besides, miR-383-5p elevated OXA sensitivity of GC cells by repressing FGF7. Moreover, the deletion of circLRCH3 increased OXA sensitivity in vivo. Knockdown of circLRCH3 alleviated OXA resistance of GC by modulating the miR-383-5p/FGF7 axis, which provided a promising therapeutic target for GC chemoresistance.

Genomic analysis in patients with soft tissue sarcoma to examine gene mutations involved in chromosome segregation, mitotic spindle assembly, and degradation.

e23523 Background: Soft tissue sarcoma (STS) is a rare cancer with few drug discovery. Recent genomic analysis of STS revealed a high degree of chromosomal instability (CIN) including genome-wide copy number alterations, aneuploidy, whole genome doubling and chromothripsis. In STS patients, we have reported extensive somatic LOH in tumors, a hallmark of CIN, represented by haploid of germline mutations/variants in cancer-related genes. CIN of STS implicates abnormalities in chromosome segregation, spindle assembly and degradation in mitosis but few studies have examined the impact of gene mutations involved in the pathological mitotic processes in STS. Methods: We recruited 155 patients with metastatic/recurrent STS (135 female and 20 male, mean age 51 at analysis, 100 LMS, 19 LPS, 4 ESS, 3 UPS, 3 AS, 3 MPT and others) with information of familial cancer burden. Whole exome sequencing and analysis were performed in both blood and tumor samples as described in 2018ASCO. Results: STS Patients with tumors harboring less than 33% somatic LOH (n = 54) in a total of somatic and LOH mutations/variants in 595 COSMIC genes have a better prognosis than those (34-66%, n = 49, 67-100%, n = 52) with more LOH genes (5-year OS; 72% vs 53% or 40%, p = 0.0499, p = 0.0023, respectively). LOH was associated with neither TMB nor MSI status but with a total number of somatic and germline mutations (R = 0.874). Of the genes involved in chromosome segregation and mitosis, we found that a family of ARHGAP genes which play a role in the spindle degradation and Aurora A kinase activation was most frequently mutated in tumor and germline genomes (n = 102 in a total of 155, n = 71 in 100 LMS). Damaging ARHGAP mutations/variants are correlated with higher LOH values (54.1±2.8%, n = 102 vs. 33.1±3.8%, n = 53, mean±SE, p < 0.001, for LMS, 63.3±2.9%, n = 71 vs. 36.7±5.4%, n = 29, mean±SE, p < 0.001) and poor prognosis of patients (5-year OS; 48% n = 102 vs. 71% n = 53, p = 0.0107). We also found damaging mutations/variants in a family of CAPN (calpain) genes involved in cleavage of the cohesion ring for chromosome separation (n = 73 in 155) and CENPT gene essential for bridging between kinetochore and microtubules via NDC80 (n = 43 in 155). For CENPT, loss of wild type allele at codons encoding functional Ser121 and Arg122 was found in 10 patients with LMS and DSRCT. Damaging CAPN mutations/variants are correlated with higher LOH values (60.6±3.0%, n = 73 vs. 34.8±3.2%, n = 82, mean±SE, p < 0.001). Conclusions: This study demonstrates extensive genomic abnormalities involved in the pathways of chromosome segregation and mitosis in STS. Recent studies, including ourselves, confirmed overexpression of KIF18A, NDC80 and NUF2 in STS, lethal targets for KIF18A and CHD (Calponin Homology Domain) inhibitors in CIN tumors. Together, our results will open a new avenue evolving treatment landscape of STS.

The WW domain of IQGAP1 binds directly to the p110α catalytic subunit of PI 3-kinase.

IQGAP1 is a multi-domain cancer-associated protein that serves as a scaffold protein for multiple signaling pathways. Numerous binding partners have been found for the calponin homology, IQ and GAP-related domains in IQGAP1. Identification of a binding partner for its WW domain has proven elusive, however, even though a cell-penetrating peptide derived from this domain has marked anti-tumor activity. Here, using in vitrobinding assays with human proteins and co-precipitation from human cells, we show that the WW domain of human IQGAP1 binds directly to the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K). In contrast, the WW domain does not bind to ERK1/2, MEK1/2, or the p85α regulatory subunit of PI3K when p85α is expressed alone. However, the WW domain is able to bind to the p110α/p85α heterodimer when both subunits are co-expressed, as well as to the mutationally activated p110α/p65α heterodimer. We present a model of the structure of the IQGAP1 WW domain, and experimentally identify key residues in the hydrophobic core and beta strands of the WW domain that are required for binding to p110α. These findings contribute to a more precise understanding of IQGAP1-mediated scaffolding, and of how IQGAP1-derived therapeutic peptides might inhibit tumorigenesis.

FHODs: Nuclear tethered formins for nuclear mechanotransduction.

In this review, we discuss FHOD formins with a focus on recent studies that reveal a new role for them as critical links for nuclear mechanotransduction. The FHOD family in vertebrates comprises two structurally related proteins, FHOD1 and FHOD3. Their similar biochemical properties suggest overlapping and redundant functions. FHOD1 is widely expressed, FHOD3 less so, with highest expression in skeletal (FHOD1) and cardiac (FHOD3) muscle where specific splice isoforms are expressed. Unlike other formins, FHODs have strong F-actin bundling activity and relatively weak actin polymerization activity. These activities are regulated by phosphorylation by ROCK and Src kinases; bundling is additionally regulated by ERK1/2 kinases. FHODs are unique among formins in their association with the nuclear envelope through direct, high affinity binding to the outer nuclear membrane proteins nesprin-1G and nesprin-2G. Recent crystallographic structures reveal an interaction between a conserved motif in one of the spectrin repeats (SRs) of nesprin-1G/2G and a site adjacent to the regulatory domain in the amino terminus of FHODs. Nesprins are components of the LINC (linker of nucleoskeleton and cytoskeleton) complex that spans both nuclear membranes and mediates bidirectional transmission of mechanical forces between the nucleus and the cytoskeleton. FHODs interact near the actin-binding calponin homology (CH) domains of nesprin-1G/2G enabling a branched connection to actin filaments that presumably strengthens the interaction. At the cellular level, the tethering of FHODs to the outer nuclear membrane mechanically couples perinuclear actin arrays to the nucleus to move and position it in fibroblasts, cardiomyocytes, and potentially other cells. FHODs also function in adhesion maturation during cell migration and in the generation of sarcomeres, activities distant from the nucleus but that are still influenced by it. Human genetic studies have identified multiple FHOD3 variants linked to dilated and hypertrophic cardiomyopathies, with many mutations mapping to "hot spots" in FHOD3 domains. We discuss how FHOD1/3's role in reinforcing the LINC complex and connecting to perinuclear actin contributes to functions of mechanically active tissues such as striated muscle.

CircSPECC1 Promotes Proliferation and Migration of LNCaP Prostate Cancer Cells by Affecting Their Epithelial-Mesenchymal Transition.

To determine the effects of circSPECC1 (hsa_circ_0000745) on the proliferation and migration of LNCaP prostate cancer cells and to explore the potential molecular mechanism. Stable circSPECC1 shRNA-expressing and circSPECC1-overexpressing LNCaP cell lines were constructed, and relative gene expression levels were determined by RT-PCR. MTT and clonogenic assays were used to assess proliferative ability while a scratch test was used to analyze cell migration. Western blotting was used to determine protein expression levels. The effects of circSPECC1 on the proliferation of LNCaP prostate cancer cells were observed in vivo. circSPECC1 was found to be derived from the SPECC1 (sperm antigen with calponin homology and coiled-coil domains 1) parent gene and to form a loop. Overexpression of circSPECC1 promoted the proliferation and migration of the LNCaP cells, whereas decreased expression of circSPECC1 inhibited these properties. Overexpression of circSPECC1 promoted the expression of MMP-2, MMP-9, VEGF, vimentin, and N-cad but downregulated the expression of E-cad. Decreased expression of circSPECC1 inhibited the expression of MMP-2, MMP-9, VEGF, vimentin, and N-cad but increased the expression of E-cad. circSPECC1 promotes cell proliferation and migration by affecting the epithelial-mesenchymal transition of LNCaP prostate cancer cells.

MFN2 deficiency affects calcium homeostasis in lung adenocarcinoma cells via downregulation of UCP4.

Mitofusin-2 (MFN2) is a transmembrane GTPase that regulates mitochondrial fusion and thereby modulates mitochondrial function. However, the role of MFN2 in lung adenocarcinoma remains controversial. Here, we investigated the effect of MFN2 regulation on mitochondria in lung adenocarcinoma. We found that MFN2 deficiency resulted in decreased UCP4 expression and mitochondrial dysfunction in A549 and H1975 cells. UCP4 overexpression restored ATP and intracellular calcium concentration, but not mtDNA copy number, mitochondrial membrane potential or reactive oxygen species level. Furthermore, mass spectrometry analysis identified 460 overlapping proteins after independent overexpression of MFN2 and UCP4; these proteins were significantly enriched in the cytoskeleton, energy production, and calponin homology (CH) domains. Moreover, the calcium signaling pathway was confirmed to be enriched in KEGG pathway analysis. We also found by protein-protein interaction network analysis that PINK1 may be a key regulator of MFN2- and UCP4-mediated calcium homeostasis. Furthermore, PINK1 increased MFN2/UCP4-mediated intracellular Ca2+ concentration in A549 and H1975 cells. Finally, we demonstrated that low expression levels of MFN2 and UCP4 in lung adenocarcinoma are associated with poor clinical prognosis. In conclusion, our data suggest not only a potential role of MFN2 and UCP4 in co-regulating calcium homeostasis in lung adenocarcinoma but also their potential use as therapeutic targets in lung cancer.

The Neuron Navigators: Structure, function, and evolutionary history.

Neuron navigators (Navigators) are cytoskeletal-associated proteins important for neuron migration, neurite growth, and axon guidance, but they also function more widely in other tissues. Recent studies have revealed novel cellular functions of Navigators such as macropinocytosis, and have implicated Navigators in human disorders of axon growth. Navigators are present in most or all bilaterian animals: vertebrates have three Navigators (NAV1-3), Drosophila has one (Sickie), and Caenorhabditis elegans has one (Unc-53). Structurally, Navigators have conserved N- and C-terminal regions each containing specific domains. The N-terminal region contains a calponin homology (CH) domain and one or more SxIP motifs, thought to interact with the actin cytoskeleton and mediate localization to microtubule plus-end binding proteins, respectively. The C-terminal region contains two coiled-coil domains, followed by a AAA+ family nucleoside triphosphatase domain of unknown activity. The Navigators appear to have evolved by fusion of N- and C-terminal region homologs present in simpler organisms. Overall, Navigators participate in the cytoskeletal response to extracellular cues via microtubules and actin filaments, in conjunction with membrane trafficking. We propose that uptake of fluid-phase cues and nutrients and/or downregulation of cell surface receptors could represent general mechanisms that explain Navigator functions. Future studies developing new models, such as conditional knockout mice or human cerebral organoids may reveal new insights into Navigator function. Importantly, further biochemical studies are needed to define the activities of the Navigator AAA+ domain, and to study potential interactions among different Navigators and their binding partners.

Placental macrophage responses to viral and bacterial ligands and the influence of fetal sex.

Bacterial and viral infections of the placenta are associated with inflammation and adverse pregnancy outcomes. Hofbauer cells (HBCs) are fetal-origin macrophages in the placenta, proposed to protect the fetus from vertical pathogen transmission. We performed quantitative proteomics on term HBCs under resting conditions and following exposure to bacterial and viral pathogen-associated molecular patterns (PAMPs), and investigated the contribution of fetal sex. Resting HBCs expressed proteins pertinent to macrophage function, including chemokines, cytokines, Toll-like receptors, and major histocompatibility complex class I and II molecules. HBCs mounted divergent responses to bacterial versus viral PAMPs but exhibited protein expression changes suggestive of a more pro-inflammatoryphenotype. A comparison between male and female HBCs showed that the latter mounted a stronger and wider response. Here, we provide a comprehensive understanding of the sex-dependent responses of placental macrophages to infectious triggers, which were primarily associated with lipid metabolism in males and cytoskeleton organization in females.

Strain stiffening of Ndc80 complexes attached to microtubule plus ends

In the mitotic spindle, microtubules attach to chromosomes via kinetochores. The microtubule-binding Ndc80 complex is an integral part of kinetochores, and is essential for kinetochores to attach to microtubules and to transmit forces from dynamic microtubule ends to the chromosomes. The Ndc80 complex has a rod-like appearance with globular domains at its ends that are separated by a long coiled coil. Its mechanical properties are considered important for the dynamic interaction between kinetochores and microtubules. Here, we present a novel method that allows us to time trace the effective stiffness of Ndc80 complexes following shortening microtubule ends against applied force in optical trap experiments. Applying this method to wild-type Ndc80 and three variants (calponin homology (CH) domains mutated or Hec1 tail unphosphorylated, phosphorylated, or truncated), we reveal that each variant exhibits strain stiffening; i.e., the effective stiffness increases under tension that is built up by a depolymerizing microtubule. The strain stiffening relation is roughly linear and independent of the state of the microtubule. We introduce structure-based models that show that the strain stiffening can be traced back to the specific architecture of the Ndc80 complex with a characteristic flexible kink, to thermal fluctuations of the microtubule, and to the bending elasticity of flaring protofilaments, which exert force to move the Ndc80 complexes. Our model accounts for changes in the amount of load-bearing attachments at various force levels and reproduces the roughly linear strain stiffening behavior, highlighting the importance of force-dependent binding affinity.

MICAL1 activation by PAK1 mediates actin filament disassembly.

The MICAL1 monooxygenase is an important regulator of filamentous actin (F-actin) structures. Although MICAL1 has been shown to be regulated via protein-protein interactions at the autoinhibitory carboxyl terminus, a link between actin-regulatory RHO GTPase signaling pathways and MICAL1 has not been established. We show that the CDC42 GTPase effector PAK1 associates with and phosphorylates MICAL1 on two serine residues, leading to accelerated F-actin disassembly. PAK1 binds to the amino-terminal catalytic monooxygenase and calponin homology domains, distinct from the autoinhibitory carboxyl terminus. Extracellular ligand stimulation leads to PAK-dependent phosphorylation, linking external signals to MICAL1 phosphorylation. Mass spectrometry indicates that MICAL1 co-expression with CDC42 and PAK1 increases MICAL1 association with hundreds of proteins, including the previously described MICAL1-interacting proteins RAB10 and RAB7A. These results provide insights into a redox-mediated pathway linking extracellular signals to cytoskeleton regulation via a RHO GTPase and indicate a means of communication between RHO and RAB GTPases.