Mechanism of actin filament severing and capping by gelsolin.

Gelsolin is the prototypical member of a family of Ca2+-activated F-actin severing and capping proteins. Here we report structures of Ca2+-bound human gelsolin at the barbed end of F-actin. One structure reveals gelsolin's six domains (G1G6) and interdomain linkers wrapping around F-actin, while another shows domains G1G3-a fragment observed during apoptosis-binding on both sides of F-actin. Conformational changes that trigger severing occur on one side of F-actin with G1G6 and on both sides with G1G3. Gelsolin remains bound after severing, blocking subunit exchange.

Clinical and genetic delineation of autosomal recessive and dominant ACTL6B-related developmental brain disorders.

PURPOSE: This study aims to comprehensively delineate the phenotypic spectrum of ACTL6B-related disorders, previously associated with both autosomal recessive and autosomal dominant neurodevelopmental disorders. Molecularly, the role of the nucleolar protein ACTL6B in contributing to the disease has remained unclear. METHODS: We identified 105 affected individuals, including 39 previously reported cases, and systematically analysed detailed clinical and genetic data for all individuals. Additionally, we conducted knockdown experiments in neuronal cells to investigate the role of ACTL6B in ribosome biogenesis. RESULTS: Biallelic variants in ACTL6B are associated with severe-to-profound global developmental delay/intellectual disability (GDD/ID), infantile intractable seizures, absent speech, autistic features, dystonia, and increased lethality. De novo monoallelic variants result in moderate-to-severe GDD/ID, absent speech, and autistic features, while seizures and dystonia were less frequently observed. Dysmorphic facial features and brain abnormalities, including hypoplastic corpus callosum, parenchymal volume loss/atrophy, are common findings in both groups. We reveal that in the nucleolus, ACTL6B plays a crucial role in ribosome biogenesis, in particular in pre-rRNA processing. CONCLUSION: This study provides a comprehensive characterization of the clinical spectrum of both autosomal recessive and dominant forms of ACTL6B-associated disorders. It offers a comparative analysis of their respective phenotypes provides a plausible molecular explanation and suggests their inclusion within the expanding category of 'ribosomopathies'.

Mechanism of Actin Filament Severing and Capping by Gelsolin.

Gelsolin is the prototypical member of a family of Ca 2+ -dependent F-actin severing and capping proteins. A structure of Ca 2+ -bound full-length gelsolin at the barbed end shows domains G1G6 and the inter-domain linkers wrapping around F-actin. Another structure shows domains G1G3, a fragment produced during apoptosis, on both sides of F-actin. Conformational changes that trigger severing occur on one side of F-actin with G1G6 and on both sides with G1G3. Gelsolin remains bound after severing, blocking subunit exchange.

Myosin-I synergizes with Arp2/3 complex to enhance the pushing forces of branched actin networks.

Class I myosins (myosin-Is) colocalize with Arp2/3 complex-nucleated actin networks at sites of membrane protrusion and invagination, but the mechanisms by which myosin-I motor activity coordinates with branched actin assembly to generate force are unknown. We mimicked the interplay of these proteins using the "comet tail" bead motility assay, where branched actin networks are nucleated by the Arp2/3 complex on the surface of beads coated with myosin-I and nucleation-promoting factor. We observed that myosin-I increased bead movement efficiency by thinning actin networks without affecting growth rates. Myosin-I triggered symmetry breaking and comet tail formation in dense networks resistant to spontaneous fracturing. Even with arrested actin assembly, myosin-I alone could break the network. Computational modeling recapitulated these observations, suggesting myosin-I acts as a repulsive force shaping the network's architecture and boosting its force-generating capacity. We propose that myosin-I leverages its power stroke to amplify the forces generated by Arp2/3 complex-nucleated actin networks.

Myosin-1C differentially displaces tropomyosin isoforms altering their inhibition of motility.

Force generation and motility by actomyosin in nonmuscle cells are spatially regulated by ∼40 tropomyosin (Tpm) isoforms. The means by which Tpms are targeted to specific cellular regions and the mechanisms that result in differential activity of myosin paralogs are unknown. We show that Tpm3.1 and Tpm1.7 inhibit Myosin-IC (Myo1C), with Tpm1.7 more effectively reducing the number of gliding filaments than Tpm3.1. Strikingly, cosedimentation and fluorescence microscopy assays revealed that Tpm3.1 is displaced from actin by Myo1C and not by myosin-II. In contrast, Tpm1.7 is only weakly displaced by Myo1C. Unlike other characterized myosins, Myo1C motility is inhibited by Tpm when the Tpm-actin filament is activated by myosin-II. These results point to a mechanism for the exclusion of myosin-I paralogs from cellular Tpm-decorated actin filaments that are activated by other myosins. Additionally, our results suggest a potential mechanism for myosin-induced Tpm sorting in cells.

Mechanisms of actin filament severing and elongation by formins.

Humans express 15 formins that play crucial roles in actin-based processes, including cytokinesis, cell motility and mechanotransduction1,2. However, the lack of structures bound to the actin filament (F-actin) has been a major impediment to understanding formin function. Whereas formins are known for their ability to nucleate and elongate F-actin3-7, some formins can additionally depolymerize, sever or bundle F-actin. Two mammalian formins, inverted formin 2 (INF2) and diaphanous 1 (DIA1, encoded by DIAPH1), exemplify this diversity. INF2 shows potent severing activity but elongates weakly8-11 whereas DIA1 has potent elongation activity but does not sever4,8. Using cryo-electron microscopy (cryo-EM) we show five structural states of INF2 and two of DIA1 bound to the middle and barbed end of F-actin. INF2 and DIA1 bind differently to these sites, consistent with their distinct activities. The formin-homology 2 and Wiskott-Aldrich syndrome protein-homology 2 (FH2 and WH2, respectively) domains of INF2 are positioned to sever F-actin, whereas DIA1 appears unsuited for severing. These structures also show how profilin-actin is delivered to the fast-growing barbed end, and how this is followed by a transition of the incoming monomer into the F-actin conformation and the release of profilin. Combined, the seven structures presented here provide step-by-step visualization of the mechanisms of F-actin severing and elongation by formins.

Pharmacologic blockade of nicotinic receptors in the suprachiasmatic nucleus increases ovarian atresia and inhibits follicular growth.

Reproduction in all mammalian species depends on the growth and maturation of ovarian follicles, that is, folliculogenesis. Follicular development can culminate with the rupture of mature follicles and the consequent expulsion of their oocytes (ovulation) or in atresia, characterized by the arrest of development and eventual degeneration. These processes are regulated by different neuroendocrine signals arising at different hypothalamic nuclei, including the suprachiasmatic nucleus (SCN). In the later, the activation of muscarinic receptors (mAChRs) and nicotinic receptors (nAChRs) by acetylcholine is essential for the regulation of the pre-ovulatory signals that stimulate the rupture of mature follicles. To evaluate the participation of the nAChRs in the SCN throughout the oestrous cycle in the regulation of the hypothalamic-pituitary-ovarian axis. For this purpose, 90-day-old adult female rats in metoestrus, dioestrus, proestrus or oestrus were microinjected into the left- or right-SCN with 0.3 µL of saline solution as vehicle or with 0.225 µg of mecamylamine (Mec), a non-selective antagonist of the nicotinic receptors, diluted in 0.3 µL of vehicle. The animals were sacrificed when they presented vaginal cornification, indicative of oestrus stage, and the effects of the unilateral pharmacological blockade of the nAChRs in the SCN on follicular development, ovulation and secretion of oestradiol and follicle-stimulating hormone (FSH) were evaluated. The microinjection of Mec decreased the serum levels of FSH, which resulted in a lower number of growing and healthy follicles and an increase in atresia. The higher percentage of atresia in pre-ovulatory follicles was related to a decrease in the number of ova shed and abnormalities in oestradiol secretion. We also detected asymmetric responses between the left and right treatments that depended on the stage of the oestrous cycle. The present results allow us to suggest that during all the stages of the oestrous cycle, cholinergic signals that act on the nAChRs in the SCN are pivotal to modulate the secretion of gonadotropins and hence the physiology of the ovaries. Further research is needed to determine if such signals are generated by the cholinergic neurons in the SCN or by cholinergic afferents to the SCN.

Molecular mechanisms linking missense ACTG2 mutations to visceral myopathy.

Visceral myopathy is a life-threatening disease characterized by muscle weakness in the bowel, bladder, and uterus. Mutations in smooth muscle γ-actin (ACTG2) are the most common cause of the disease, but the mechanisms by which the mutations alter muscle function are unknown. Here, we examined four prevalent ACTG2 mutations (R40C, R148C, R178C, and R257C) that cause different disease severity and are spread throughout the actin fold. R178C displayed premature degradation, R148C disrupted interactions with actin-binding proteins, R40C inhibited polymerization, and R257C destabilized filaments. Because these mutations are heterozygous, we also analyzed 50/50 mixtures with wild-type (WT) ACTG2. The WT/R40C mixture impaired filament nucleation by leiomodin 1, and WT/R257C produced filaments that were easily fragmented by smooth muscle myosin. Smooth muscle tropomyosin isoform Tpm1.4 partially rescued the defects of R40C and R257C. Cryo-electron microscopy structures of filaments formed by R40C and R257C revealed disrupted intersubunit contacts. The biochemical and structural properties of the mutants correlate with their genotype-specific disease severity.

Optical Control of G-Actin with a Photoswitchable Latrunculin.

Actin is one of the most abundant proteins in eukaryotic cells and is a key component of the cytoskeleton. A range of small molecules has emerged that interfere with actin dynamics by either binding to polymeric F-actin or monomeric G-actin to stabilize or destabilize filaments or prevent their formation and growth, respectively. Among these, the latrunculins, which bind to G-actin and affect polymerization, are widely used as tools to investigate actin-dependent cellular processes. Here, we report a photoswitchable version of latrunculin, termed opto-latrunculin (OptoLat), which binds to G-actin in a light-dependent fashion and affords optical control over actin polymerization. OptoLat can be activated with 390-490 nm pulsed light and rapidly relaxes to its inactive form in the dark. Light activated OptoLat induced depolymerization of F-actin networks in oligodendrocytes and budding yeast, as shown by fluorescence microscopy. Subcellular control of actin dynamics in human cancer cell lines was demonstrated via live cell imaging. Light-activated OptoLat also reduced microglia surveillance in organotypic mouse brain slices while ramification was not affected. Incubation in the dark did not alter the structural and functional integrity of the microglia. Together, our data demonstrate that OptoLat is a useful tool for the elucidation of G-actin dependent dynamic processes in cells and tissues.

Myosin-I Synergizes with Arp2/3 Complex to Enhance Pushing Forces of Branched Actin Networks.

Myosin-Is colocalize with Arp2/3 complex-nucleated actin networks at sites of membrane protrusion and invagination, but the mechanisms by which myosin-I motor activity coordinates with branched actin assembly to generate force are unknown. We mimicked the interplay of these proteins using the "comet tail" bead motility assay, where branched actin networks are nucleated by Arp2/3 complex on the surface of beads coated with myosin-I and the WCA domain of N-WASP. We observed that myosin-I increased bead movement efficiency by thinning actin networks without affecting growth rates. Remarkably, myosin-I triggered symmetry breaking and comet-tail formation in dense networks resistant to spontaneous fracturing. Even with arrested actin assembly, myosin-I alone could break the network. Computational modeling recapitulated these observations suggesting myosin-I acts as a repulsive force shaping the network's architecture and boosting its force-generating capacity. We propose that myosin-I leverages its power stroke to amplify the forces generated by Arp2/3 complex-nucleated actin networks.

The differential sensitivity of the hypothalamic-hypophysial-ovarian axis to 5-hydroxytryptophan alters the secretion of estradiol.

Serotonin [5-hydroxytryptamine (5-HT)] modulates ovarian function. The precursor of 5-HT, 5-hydroxytryptophan (5-HTP), has been used to treat depression. However, the effects of 5-HTP on ovarian and reproductive physiology remain unknown. In this research, we analysed the impact of 5-HTP on the monoaminergic system and its interactions with the reproductive axis and ovarian estradiol secretion when administered by distinct routes. Female rats 30 days of age were injected with 5-HTP i.p. (100 mg/kg), into the ovarian bursa (1.5 µg/40 µL) or into the median raphe nucleus (20 µg/2.5 µL) and were killed 60 or 120 min after injection. As controls, we used rats of the same age injected with vehicle (0.9% NaCl). Monoamine, gonadotrophin and steroid ovarian hormone concentrations were measured. The injection of 5-HTP either i.p. or directly into the ovarian bursa increased the concentrations of 5-HT and the metabolite 5-hydroxyindole-3-acetic acid in the ovary. For both routes of administration, the serum concentration of estradiol increased. After i.p. injection of 5-HTP, the concentrations of luteinizing hormone were decreased and follicle-stimulating hormone increased after 120 min. Micro-injection of 5-HTP into the median raphe nucleus increased the concentrations of 5-HT in the anterior hypothalamus and dopamine in the medial hypothalamus after 120 min. Our results suggest that the administration of 5-HTP either i.p. or directly into the ovarian bursa enhances ovarian estradiol secretion.

Interaction between the mitochondrial adaptor MIRO and the motor adaptor TRAK.

MIRO (mitochondrial Rho GTPase) consists of two GTPase domains flanking two Ca2+-binding EF-hand domains. A C-terminal transmembrane helix anchors MIRO to the outer mitochondrial membrane, where it functions as a general adaptor for the recruitment of cytoskeletal proteins that control mitochondrial dynamics. One protein recruited by MIRO is TRAK (trafficking kinesin-binding protein), which in turn recruits the microtubule-based motors kinesin-1 and dynein-dynactin. The mechanism by which MIRO interacts with TRAK is not well understood. Here, we map and quantitatively characterize the interaction of human MIRO1 and TRAK1 and test its potential regulation by Ca2+ and/or GTP binding. TRAK1 binds MIRO1 with low micromolar affinity. The interaction was mapped to a fragment comprising MIRO1's EF-hands and C-terminal GTPase domain and to a conserved sequence motif within TRAK1 residues 394 to 431, immediately C-terminal to the Spindly motif. This sequence is sufficient for MIRO1 binding in vitro and is necessary for MIRO1-dependent localization of TRAK1 to mitochondria in cells. MIRO1's EF-hands bind Ca2+ with dissociation constants (KD) of 3.9 µM and 300 nM. This suggests that under cellular conditions one EF-hand may be constitutively bound to Ca2+ whereas the other EF-hand binds Ca2+ in a regulated manner, depending on its local concentration. Yet, the MIRO1-TRAK1 interaction is independent of Ca2+ binding to the EF-hands and of the nucleotide state (GDP or GTP) of the C-terminal GTPase. The interaction is also independent of TRAK1 dimerization, such that a TRAK1 dimer can be expected to bind two MIRO1 molecules on the mitochondrial surface.

Unilateral lesion of the suprachiasmatic nucleus impairs estradiol feedback, follicular development, estrous cycle and ovulation.

In brief: The SCN regulates ovulation by stimulating the preovulatory surge of gonadotropins. This study revealed an additional role in the sensitization of the hypothalamus to estradiol that changes along the estrous cycle and the side of the nucleus. Abstract: Ovulation is timed by neural signals originating at the suprachiasmatic nucleus (SCN) that trigger ovulation when converge with high estradiol levels, which indicates the maturation of ovarian follicles. We have shown that the hypothalamic regulation of ovulation is asymmetrical and we hypothesized that the paired SCN could contribute to such symmetries. We unilaterally lesioned the SCN of rats at each stage of the estrous cycle and evaluated the acute effects on the progression of their estrous cycle, follicular development and ovulation. Lesions prevented progression of the estrous cycle when performed in estrus/metestrus but not in diestrus/proestrus. Abnormalities in follicular development were observed in the nonovulating lesioned rats and this was independent of the side of the SCN destroyed and the stage of the cycle when surgery was performed. Groups of lesioned rats were then hormonally primed with GnRH or estradiol to assess the neuroendocrine pathway altered by the treatment. GnRH restored ovulation, suggesting that both SCN are needed for proper triggering of the preovulatory surge of GnRH and that unilateral lesion does not impair the sensitivity of the pituitary or the ovary to GnRH and gonadotropins, respectively. With regard to restoring ovulation, estradiol was asymmetrically effective in rats lesioned in estrous, partially effective in rats operated at diestrus and ineffective in rats at metestrus. Our results indicate that the SCN regulates the activity of the hypothalamic-pituitary-ovarian axis not only by modulating the preovulatory surge of GnRH/gonadotropins but also by promoting the hypothalamic integration of estrogenic signals from the ovaries in an asymmetric and stage-dependent fashion.

Mechanism of synergistic activation of Arp2/3 complex by cortactin and WASP-family proteins.

Cortactin coactivates Arp2/3 complex synergistically with WASP-family nucleation-promoting factors (NPFs) and stabilizes branched networks by linking Arp2/3 complex to F-actin. It is poorly understood how cortactin performs these functions. We describe the 2.89 Å resolution cryo-EM structure of cortactin's N-terminal domain (Cort1-76) bound to Arp2/3 complex. Cortactin binds Arp2/3 complex through an inverted Acidic domain (D20-V29), which targets the same site on Arp3 as the Acidic domain of NPFs but with opposite polarity. Sequences N- and C-terminal to cortactin's Acidic domain do not increase its affinity for Arp2/3 complex but contribute toward coactivation with NPFs. Coactivation further increases with NPF dimerization and for longer cortactin constructs with stronger binding to F-actin. The results suggest that cortactin contributes to Arp2/3 complex coactivation with NPFs in two ways, by helping recruit the complex to F-actin and by stabilizing the short-pitch (active) conformation, which are both byproducts of cortactin's core function in branch stabilization.

Transition State of Arp2/3 Complex Activation by Actin-Bound Dimeric Nucleation-Promoting Factor.

Arp2/3 complex generates branched actin networks that drive fundamental processes such as cell motility and cytokinesis. The complex comprises seven proteins, including actin-related proteins (Arps) 2 and 3 and five scaffolding proteins (ArpC1-ArpC5) that mediate interactions with a pre-existing (mother) actin filament at the branch junction. Arp2/3 complex exists in two main conformations, inactive with the Arps interacting end-to-end and active with the Arps interacting side-by-side like subunits of the short-pitch helix of the actin filament. Several cofactors drive the transition toward the active state, including ATP binding to the Arps, WASP-family nucleation-promoting factors (NPFs), actin monomers, and binding of Arp2/3 complex to the mother filament. The precise contribution of each cofactor to activation is poorly understood. We report the 3.32-Å resolution cryo-electron microscopy structure of a transition state of Arp2/3 complex activation with bound constitutively dimeric NPF. Arp2/3 complex-binding region of the NPF N-WASP was fused C-terminally to the α and ß subunits of the CapZ heterodimer. One arm of the NPF dimer binds Arp2 and the other binds actin and Arp3. The conformation of the complex is intermediate between those of inactive and active Arp2/3 complex. Arp2, Arp3, and actin also adopt intermediate conformations between monomeric (G-actin) and filamentous (F-actin) states, but only actin hydrolyzes ATP. In solution, the transition complex is kinetically shifted toward the short-pitch conformation and has higher affinity for F-actin than inactive Arp2/3 complex. The results reveal how all the activating cofactors contribute in a coordinated manner toward Arp2/3 complex activation.

Conformation of actin subunits at the barbed and pointed ends of F-actin with and without capping proteins.

Advances in cryo-electron microscopy have made possible the determination of structures of the barbed and pointed ends of F-actin, both in the absence and the presence of capping proteins that block subunit exchange. The conformation of the two exposed protomers at the barbed end resembles the "flat" conformation of protomers in the middle of F-actin. The barbed end changes little upon binding of CapZ, which in turn undergoes a major conformational change. At the pointed end, however, protomers have the "twisted" conformation characteristic of G-actin, whereas tropomodulin binding forces a flat conformation upon the second subunit. The structures provide a mechanistic understanding for the asymmetric addition/dissociation of actin subunits at the ends of F-actin and open the way to future studies of other regulators of filament end dynamics.

Structures of the free and capped ends of the actin filament.

The barbed and pointed ends of the actin filament (F-actin) are the sites of growth and shrinkage and the targets of capping proteins that block subunit exchange, including CapZ at the barbed end and tropomodulin at the pointed end. We describe cryo-electron microscopy structures of the free and capped ends of F-actin. Terminal subunits at the free barbed end adopt a "flat" F-actin conformation. CapZ binds with minor changes to the barbed end but with major changes to itself. By contrast, subunits at the free pointed end adopt a "twisted" monomeric actin (G-actin) conformation. Tropomodulin binding forces the second subunit into an F-actin conformation. The structures reveal how the ends differ from the middle in F-actin and how these differences control subunit addition, dissociation, capping, and interactions with end-binding proteins.

Single particle cryo-EM analysis of Rickettsia conorii Sca2 reveals a formin-like core.

Spotted fever group Rickettsia undergo actin-based motility inside infected eukaryotic cells using Sca2 (surface cell antigen 2): an âˆ¼ 1800 amino-acid monomeric autotransporter protein that is surface-attached to the bacterium and responsible for the assembly of long unbranched actin tails. Sca2 is the only known functional mimic of eukaryotic formins, yet it shares no sequence similarities to the latter. Using structural and biochemical approaches we have previously shown that Sca2 uses a novel actin assembly mechanism. The first âˆ¼ 400 amino acids fold into helix-loop-helix repeats that form a crescent shape reminiscent of a formin FH2 monomer. Additionally, the N- and C- terminal halves of Sca2 display intramolecular interaction in an end-to-end manner and cooperate for actin assembly, mimicking a formin FH2 dimer. Towards a better structural understanding of this mechanism, we performed single-particle cryo-electron microscopy analysis of Sca2. While high-resolution structural details remain elusive, our model confirms the presence of a formin-like core: Sca2 indeed forms a doughnut shape, similar in diameter to a formin FH2 dimer and can accommodate two actin subunits. Extra electron density, thought to be contributed by the C-terminal repeat domain (CRD), covering one side is also observed. This structural analysis allows us to propose an updated model where nucleation proceeds by encircling two actin subunits, and elongation proceeds either by a formin-like mechanism that necessitates conformational changes in the observed Sca2 model, or via an insertional mechanism akin to that observed in the ParMRC system.

Effects of chronic exposure to cold stress on ovarian functions in prepubertal rats.

Ovarian functions are modulated by the hypothalamus-pituitary-ovary axis and neural signals. Stress modifies the activity of the sympathetic nervous system. In adult female rats, cold stress results in higher noradrenergic and steroidogenic activity of the ovary, anovulation and the presence of ovarian cysts; however, it is unknown whether this response occurs in prepubertal rats. The purpose of this study was to analyse the effects of cold stress initiated in the prepubertal stage of female rats on ovarian function. Female rats 24 days old were exposed to three, five or eight weeks of cold stress. Autopsies were performed at the end of each stress period. The parameters analysed were the number of ova shed by ovulating animals; the number of ovulating animals; the serum concentrations of progesterone, testosterone, and oestradiol; and the ovarian concentrations of norepinephrine and 3-methoxy-4-hydroxyphenyl-glycol. Our results show that chronic cold stress applied to prepubertal rats did not modify the number of ovulating animals, the total number of ova shed, or progesterone and testosterone concentrations in any of the periods analysed. Oestradiol concentration was lower in the animals exposed to five or eight weeks of stress. The ovarian norepinephrine concentration was higher in the animals exposed to three weeks of stress and was lower at eight weeks of stress. No changes in ovarian morphology were observed. Our data suggest that the changes in noradrenergic activity resulting from chronic cold stress experienced in the prepubertal stage do not modify ovarian architecture or affect the ovulatory response in adulthood.

Prevalencia de obesidad posCovid-19 en jóvenes universitarios de Tabasco.

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