scholarly journals Structural basis for tunable control of actin dynamics by myosin-15 in mechanosensory stereocilia

2021 ◽  
Author(s):  
Rui Gong ◽  
Fangfang Jiang ◽  
Zane G Moreland ◽  
Matthew J Reynolds ◽  
Santiago Espinosa de los Reyes ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Actin Polymerization ◽  
Actin Dynamics ◽  
Structural Basis ◽  
Dependent Manner ◽  
Actin Assembly ◽  
Progressive Hearing Loss ◽  
State Dependent ◽  
Profound Deafness ◽  
D Loop

The motor protein myosin-15 is necessary for the development and maintenance of mechanosensory stereocilia, and myosin-15 mutations cause profound deafness. In a companion study, we report that myosin-15 nucleates actin filament ("F-actin") assembly and identify a progressive hearing loss mutation (p.D1647G, "jordan") which disrupts stereocilia elongation by inhibiting actin polymerization. Here, we present cryo-EM structures of myosin-15 bound to F-actin, providing a framework for interpreting deafness mutations and their impacts on myosin-stimulated actin assembly. Rigor myosin-15 evokes conformational changes in F-actin yet maintains flexibility in actin's D-loop, which mediates inter-subunit contacts, while the jordan mutant locks the D-loop in a single conformation. ADP-bound myosin-15 also locks the D-loop, which correspondingly blunts actin-polymerization stimulation. We propose myosin-15 enhances polymerization by bridging actin protomers, regulating nucleation efficiency by modulating actin's structural plasticity in a myosin nucleotide-state dependent manner. This tunable regulation of actin polymerization could be harnessed to precisely control stereocilium height.

scholarly journals Mechanical stimulation induces formin-dependent assembly of a perinuclear actin rim

2015 ◽  
Vol 112 (20) ◽  
pp. E2595-E2601 ◽  
Author(s):  
Xiaowei Shao ◽  
Qingsen Li ◽  
Alex Mogilner ◽  
Alexander D. Bershadsky ◽  
G. V. Shivashankar
Keyword(s):  
Actin Polymerization ◽  
Genetic Diseases ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Actin Dynamics ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Mechanical Stimuli ◽  
Actin Assembly ◽  
Force Application

Cells constantly sense and respond to mechanical signals by reorganizing their actin cytoskeleton. Although a number of studies have explored the effects of mechanical stimuli on actin dynamics, the immediate response of actin after force application has not been studied. We designed a method to monitor the spatiotemporal reorganization of actin after cell stimulation by local force application. We found that force could induce transient actin accumulation in the perinuclear region within ∼2 min. This actin reorganization was triggered by an intracellular Ca2+ burst induced by force application. Treatment with the calcium ionophore A23187 recapitulated the force-induced perinuclear actin remodeling. Blocking of actin polymerization abolished this process. Overexpression of Klarsicht, ANC-1, Syne Homology (KASH) domain to displace nesprins from the nuclear envelope did not abolish Ca2+-dependent perinuclear actin assembly. However, the endoplasmic reticulum- and nuclear membrane-associated inverted formin-2 (INF2), a potent actin polymerization activator (mutations of which are associated with several genetic diseases), was found to be important for perinuclear actin assembly. The perinuclear actin rim structure colocalized with INF2 on stimulation, and INF2 depletion resulted in attenuation of the rim formation. Our study suggests that cells can respond rapidly to external force by remodeling perinuclear actin in a unique Ca2+- and INF2-dependent manner.

Impaired Sphingosine-1-Phosphate Synthesis Induces Preeclampsia by Deactivating Trophoblastic YAP (Yes-Associated Protein) Through S1PR2 (Sphingosine-1-Phosphate Receptor-2)-Induced Actin Polymerizations

Hypertension ◽  
2021 ◽  
Author(s):  
Jiujiang Liao ◽  
Yangxi Zheng ◽  
Mingyu Hu ◽  
Ping Xu ◽  
Li Lin ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Sphingosine Kinase ◽  
Actin Dynamics ◽  
Extravillous Trophoblast ◽  
Trophoblast Invasion ◽  
Hippo Signaling ◽  
Dependent Manner ◽  
Sphingosine Kinase 1 ◽  
Sphingosine 1 Phosphate

Incomplete spiral artery remodeling, caused by impaired extravillous trophoblast invasion, is a fundamental pathogenic process associated with malplacentation and the development of preeclampsia. Nevertheless, the mechanisms controlling this regulation of trophoblast invasion are largely unknown. We report that sphingosine-1-phosphate synthesis and expression is abundant in healthy trophoblast, whereas in pregnancies complicated by preeclampsia the placentae are associated with reduced sphingosine-1-phosphate and lower SPHK1 (sphingosine kinase 1) expression and activity. In vivo inhibition of sphingosine kinase 1 activity during placentation in pregnant mice led to decreased placental sphingosine-1-phosphate production and defective placentation, resulting in a preeclampsia phenotype. Moreover, sphingosine-1-phosphate increased HTR8/SVneo (immortalized trophoblast cells) cell invasion in a Hippo-signaling–dependent transcriptional coactivator YAP (Yes-associated protein) dependent manner, which is activated by S1PR2 (sphingosine-1-phosphate receptor-2) and downstream RhoA/ROCK induced actin polymerization. Mutation-based YAP-5SA demonstrated that sphingosine-1-phosphate activation of YAP could be either dependent or independent of Hippo signaling. Together, these findings suggest a novel pathogenic pathway of preeclampsia via disrupted sphingosine-1-phosphate metabolism and signaling-induced, interrupted actin dynamics and YAP deactivation; this may lead to potential novel intervention targets for the prevention and management of preeclampsia.

scholarly journals A role for PKC-ε in FcγR-mediated phagocytosis by RAW 264.7 cells

2002 ◽  
Vol 159 (6) ◽  
pp. 939-944 ◽  
Author(s):  
Elaine C. Larsen ◽  
Takehiko Ueyama ◽  
Pamela M. Brannock ◽  
Yasuhito Shirai ◽  
Naoaki Saito ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Variable Region ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Dependent Manner ◽  
Actin Assembly ◽  
Fcγ Receptor ◽  
Raw 264.7 Macrophages ◽  
Kinase C ◽  
Pkc Isoforms

Protein kinase C (PKC) plays a prominent role in immune signaling, and the paradigms for isoform selective signaling are beginning to be elucidated. Real-time microscopy was combined with molecular and biochemical approaches to demonstrate a role for PKC-ε in Fcγ receptor (FcγR)–dependent phagocytosis. RAW 264.7 macrophages were transfected with GFP-conjugated PKC isoforms, and GFP movement was followed during phagocytosis of fluorescent IgG–opsonized beads. PKC-ε, but not PKC-δ, concentrated around the beads. PKC-ε accumulation was transient; apparent as a “flash” on target ingestion. Similarly, endogenous PKC-ε was specifically recruited to the nascent phagosomes in a time-dependent manner. Overexpression of PKC-ε, but not PKC-α, PKC-δ, or PKC-γ enhanced bead uptake 1.8-fold. Additionally, the rate of phagocytosis in GFP PKC-ε expressors was twice that of cells expressing GFP PKC-δ. Expression of the regulatory domain (εRD) and the first variable region (εV1) of PKC-ε inhibited uptake, whereas the corresponding PKC-δ region had no effect. Actin polymerization was enhanced on expression of GFP PKC-ε and εRD, but decreased in cells expressing εV1, suggesting that the εRD and εV1 inhibition of phagocytosis is not due to effects on actin polymerization. These results demonstrate a role for PKC-ε in FcγR-mediated phagocytosis that is independent of its effects on actin assembly.

scholarly journals Arf6 Can Trigger Wave Regulatory Complex-Dependent Actin Assembly Independent of Arno

2020 ◽  
Vol 21 (7) ◽  
pp. 2457 ◽  
Author(s):  
Vikash Singh ◽  
Anthony C. Davidson ◽  
Peter J. Hume ◽  
Vassilis Koronakis
Keyword(s):  
Lipid Bilayers ◽  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Small Gtpase ◽  
Actin Dynamics ◽  
Actin Assembly ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Nucleotide Exchange Factor ◽  
Regulatory Complex

The small GTPase ADP-ribosylation factor 6 (Arf6) anchors at the plasma membrane to orchestrate key functions, such as membrane trafficking and regulating cortical actin cytoskeleton rearrangement. A number of studies have identified key players that interact with Arf6 to regulate actin dynamics in diverse cell processes, yet it is still unknown whether Arf6 can directly signal to the wave regulatory complex to mediate actin assembly. By reconstituting actin dynamics on supported lipid bilayers, we found that Arf6 in co-ordination with Rac1(Ras-related C3 botulinum toxin substrate 1) can directly trigger actin polymerization by recruiting wave regulatory complex components. Interestingly, we demonstrated that Arf6 triggers actin assembly at the membrane directly without recruiting the Arf guanine nucleotide exchange factor (GEF) ARNO (ARF nucleotide-binding site opener), which is able to activate Arf1 to enable WRC-dependent actin assembly. Furthermore, using labelled E. coli, we demonstrated that actin assembly by Arf6 also contributes towards efficient phagocytosis in THP-1 macrophages. Taken together, this study reveals a mechanism for Arf6-driven actin polymerization.

scholarly journals Profilin connects actin assembly with microtubule dynamics

2016 ◽  
Vol 27 (15) ◽  
pp. 2381-2393 ◽  
Author(s):  
Michaela Nejedla ◽  
Sara Sadi ◽  
Vadym Sulimenko ◽  
Francisca Nunes de Almeida ◽  
Hans Blom ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Actin Dynamics ◽  
Control Element ◽  
Actin Assembly ◽  
Actin Nucleation ◽  
Superresolution Microscopy ◽  
Drug Treatments ◽  
And Migration ◽  
Wasp Family Proteins ◽  
Microscopy Techniques

Profilin controls actin nucleation and assembly processes in eukaryotic cells. Actin nucleation and elongation promoting factors (NEPFs) such as Ena/VASP, formins, and WASP-family proteins recruit profilin:actin for filament formation. Some of these are found to be microtubule associated, making actin polymerization from microtubule-associated platforms possible. Microtubules are implicated in focal adhesion turnover, cell polarity establishment, and migration, illustrating the coupling between actin and microtubule systems. Here we demonstrate that profilin is functionally linked to microtubules with formins and point to formins as major mediators of this association. To reach this conclusion, we combined different fluorescence microscopy techniques, including superresolution microscopy, with siRNA modulation of profilin expression and drug treatments to interfere with actin dynamics. Our studies show that profilin dynamically associates with microtubules and this fraction of profilin contributes to balance actin assembly during homeostatic cell growth and affects micro­tubule dynamics. Hence profilin functions as a regulator of microtubule (+)-end turnover in addition to being an actin control element.

scholarly journals The Diaphanous-related Formin mDia1 Controls Serum Response Factor Activity through its Effects on Actin Polymerization

2002 ◽  
Vol 13 (11) ◽  
pp. 4088-4099 ◽  
Author(s):  
John W. Copeland ◽  
Richard Treisman
Keyword(s):  
Actin Polymerization ◽  
Serum Response Factor ◽  
Signal Pathway ◽  
Small Gtpase ◽  
Actin Dynamics ◽  
Actin Assembly ◽  
Extracellular Signals ◽  
Activation Assay ◽  
The Relationship ◽  
Serum Response

SRF-dependent transcription is regulated by the small GTPase RhoA via its effects on actin dynamics. The diaphanous-related formin (DRF) proteins have been identified as candidate RhoA effectors mediating signaling to SRF. Here we investigate the relationship between SRF activation and actin polymerization by the DRF mDia1. We show that the ability of mDia1 to potentiate SRF activity is strictly correlated with its ability to promote F-actin assembly. Both processes can occur independently of the mDia1 FH1 domain but require sequences in an extended C-terminal region encompassing the conserved FH2 domain. mDia-mediated SRF activation, but not F-actin assembly, can be blocked by a nonpolymerizable actin mutant, placing actin downstream of mDia in the signal pathway. The SRF activation assay was used to identify inactive mDia1 derivatives that inhibit serum- and LPA-induced signaling to SRF. We show that these interfering mutants also block F-actin assembly, whether induced by mDia proteins or extracellular signals. These results identify novel functional elements of mDia1 and show that it regulates SRF activity by inducing depletion of the cellular pool of G-actin.

scholarly journals Interaction between distinct actin pools controls activity-dependent actin dynamics in the dendritic spine

2016 ◽  
Author(s):  
Oleg O. Glebov ◽  
Juan Burrone
Keyword(s):  
Cell Line ◽  
Dendritic Spine ◽  
Actin Polymerization ◽  
Actin Dynamics ◽  
List Type ◽  
Dependent Manner ◽  
Complex Activity ◽  
Actin Depolymerization ◽  
Labile Pool

AbstractActin cytoskeleton is composed of functionally distinct pools of filamentous (F)-actin defined by their regulatory machinery and dynamics. Although these networks may compete for actin monomers and regulatory factors1–4, the interaction between them remains poorly understood. Here, we show that disruption of the labile F-actin pool in neurons by limited actin depolymerization5,6 unexpectedly triggers rapid enhancement of the F-actin content at the dendritic spine. Long-term blockade of NMDA-type receptors decreases spine actin polymerization, which is specifically restored by the labile pool ablation. Increase in the spine actin is triggered by blockade of formin-induced actin polymerization in a manner dependent on Arp2/3 complex activity. Finally, limited actin depolymerization increases F-actin levels in a cultured cell line, suggesting the generality of the two-tiered actin dynamics. Based on these findings, we propose a model whereby the labile pool of F-actin controlled by formin restricts the polymerization state of the Arp2/3-regulated stable spine actin, suggesting a feedback principle at the core of cytoskeletal organization in neurons.HighlightsDisruption of labile F-actin by limited depolymerization rapidly increases the synaptic F-actin content;The depolymerization-induced F-actin boost reverses decrease in synaptic F-actin induced by long-term NMDA receptor blockade;Blockade of formin-dependent actin polymerization boosts synaptic F-actin in an Arp2/3-dependent manner;Limited actin depolymerization enhances overall F-actin content in a mammalian cell line.

scholarly journals The propofol binding sites of prokaryotic voltage-gated sodium channels

2020 ◽  
Author(s):  
Elaine Yang ◽  
Weiming Bu ◽  
Antonio Suma ◽  
Vincenzo Carnevale ◽  
Kimberly C. Grasty ◽  
...  
Keyword(s):  
Binding Sites ◽  
Structural Basis ◽  
Dependent Manner ◽  
General Anesthetics ◽  
Neuronal Function ◽  
Voltage Sensors ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
State Dependent ◽  
Dynamics Simulations

AbstractPropofol, one of the most commonly used intravenous general anesthetics, modulates neuronal function by interacting with ion channels. The mechanisms that link propofol binding to the modulation of distinct ion channel states, however, are not understood. To tackle this problem, we investigated prokaryotic ancestors of eukaryotic voltage-gated Na+ channels (Navs) using unbiased photoaffinity labeling with a photoacitivatable propofol analog (AziPm), electrophysiological methods and mutagenesis. The results directly demonstrate conserved propofol binding sites involving the S4 voltage sensors and the S4-S5 linkers in NaChBac and NavMs, and also suggest state-dependent changes at these sites. Then, using molecular dynamics simulations to elucidate the structural basis of propofol modulation, we show that the S4 voltage sensors and the S4-S5 linkers shape two distinct propofol binding sites in a conformation-dependent manner. These interactions help explain how propofol binding promotes activation-coupled inactivation to inhibit Nav channel function.

scholarly journals ALS-linked PFN1 variants exhibit loss and gain of functions in the context of formin-induced actin polymerization

2021 ◽  
Vol 118 (23) ◽  
pp. e2024605118
Author(s):  
Eric J. Schmidt ◽  
Salome Funes ◽  
Jeanne E. McKeon ◽  
Brittany R. Morgan ◽  
Sivakumar Boopathy ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Actin Polymerization ◽  
Alpha Helix ◽  
Actin Dynamics ◽  
Internal Dynamic ◽  
Actin Assembly ◽  
Loss Of Function ◽  
Dynamics Simulations ◽  
Induced Changes ◽  
Lateral Sclerosis

Profilin-1 (PFN1) plays important roles in modulating actin dynamics through binding both monomeric actin and proteins enriched with polyproline motifs. Mutations in PFN1 have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, whether ALS-linked mutations affect PFN1 function has remained unclear. To address this question, we employed an unbiased proteomics analysis in mammalian cells to identify proteins that differentially interact with mutant and wild-type (WT) PFN1. These studies uncovered differential binding between two ALS-linked PFN1 variants, G118V and M114T, and select formin proteins. Furthermore, both variants augmented formin-mediated actin assembly relative to PFN1 WT. Molecular dynamics simulations revealed mutation-induced changes in the internal dynamic couplings within an alpha helix of PFN1 that directly contacts both actin and polyproline, as well as structural fluctuations within the actin- and polyproline-binding regions of PFN1. These data indicate that ALS-PFN1 variants have the potential for heightened flexibility in the context of the ternary actin–PFN1–polyproline complex during actin assembly. Conversely, PFN1 C71G was more severely destabilized than the other PFN1 variants, resulting in reduced protein expression in both transfected and ALS patient lymphoblast cell lines. Moreover, this variant exhibited loss-of-function phenotypes in the context of actin assembly. Perturbations in actin dynamics and assembly can therefore result from ALS-linked mutations in PFN1. However, ALS-PFN1 variants may dysregulate actin polymerization through different mechanisms that depend upon the solubility and stability of the mutant protein.

scholarly journals Half Way to Hypusine—Structural Basis for Substrate Recognition by Human Deoxyhypusine Synthase

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 522 ◽  
Author(s):  
Elżbieta Wątor ◽  
Piotr Wilk ◽  
Przemysław Grudnik
Keyword(s):  
Conformational Changes ◽  
Bound States ◽  
Substrate Recognition ◽  
Structural Basis ◽  
Dependent Manner ◽  
Post Translational Modification ◽  
Deoxyhypusine Synthase ◽  
Recognition Mechanism ◽  
Eukaryotic Translation ◽  
Potential Applications

Deoxyhypusine synthase (DHS) is a transferase enabling the formation of deoxyhypusine, which is the first, rate-limiting step of a unique post-translational modification: hypusination. DHS catalyses the transfer of a 4-aminobutyl moiety of polyamine spermidine to a specific lysine of eukaryotic translation factor 5A (eIF5A) precursor in a nicotinamide adenine dinucleotide (NAD)-dependent manner. This modification occurs exclusively on one protein, eIF5A, and it is essential for cell proliferation. Malfunctions of the hypusination pathway, including those caused by mutations within the DHS encoding gene, are associated with conditions such as cancer or neurodegeneration. Here, we present a series of high-resolution crystal structures of human DHS. Structures were determined as the apoprotein, as well as ligand-bound states at high-resolutions ranging from 1.41 to 1.69 Å. By solving DHS in complex with its natural substrate spermidine (SPD), we identified the mode of substrate recognition. We also observed that other polyamines, namely spermine (SPM) and putrescine, bind DHS in a similar manner as SPD. Moreover, we performed activity assays showing that SPM could to some extent serve as an alternative DHS substrate. In contrast to previous studies, we demonstrate that no conformational changes occur in the DHS structure upon spermidine-binding. By combining mutagenesis and a light-scattering approach, we show that a conserved “ball-and-chain” motif is indispensable to assembling a functional DHS tetramer. Our study substantially advances our knowledge of the substrate recognition mechanism by DHS and may aid the design of pharmacological compounds for potential applications in cancer therapy.

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