Depolymerization Of Actin Filaments Research Articles

Artificial Water Channel Promoting Depolymerization of Actin Filaments to Trigger Cancer Cell Apoptosis

Comprehensive SummaryActin filaments play important physiological functions, which have become potential targets of antitumor drugs. Using chemicals to intervene their polymerization‐depolymerization dynamics would generate new strategies for designing antitumor drugs. In this report, an artificial water channel appending acetazolamide moiety, a ligand that can selectively bind to carbonic anhydrase IX, has been prepared. We demonstrated that this conjugate can target colorectal cancer cells overexpressing carbonic anhydrase IX and trigger the depolymerization of actin filaments of the cancer cells by selectively mediating water transmembrane transport. Moreover, the conjugate‐promoted actin depolymerization led to tumor cell apoptosis and its high antitumor activity in vitro and in vivo against colorectal cancer. The method described herein represents a new and general strategy for designing antitumor drugs by using artificial channel‐mediated selective water transport to promote actin depolymerization.

Distinct functional constraints driving conservation of the cofilin N-terminal regulatory tail

Cofilin family proteins have essential roles in remodeling the cytoskeleton through filamentous actin depolymerization and severing. The short, unstructured N-terminal region of cofilin is critical for actin binding and harbors the major site of inhibitory phosphorylation. Atypically for a disordered sequence, the N-terminal region is highly conserved, but specific aspects driving this conservation are unclear. Here, we screen a library of 16,000 human cofilin N-terminal sequence variants for their capacity to support growth in S. cerevisiae in the presence or absence of the upstream regulator LIM kinase. Results from the screen and biochemical analysis of individual variants reveal distinct sequence requirements for actin binding and regulation by LIM kinase. LIM kinase recognition only partly explains sequence constraints on phosphoregulation, which are instead driven to a large extent by the capacity for phosphorylation to inactivate cofilin. We find loose sequence requirements for actin binding and phosphoinhibition, but collectively they restrict the N-terminus to sequences found in natural cofilins. Our results illustrate how a phosphorylation site can balance potentially competing sequence requirements for function and regulation.

Antibody surface mobility amplifies FcγR signaling via Arp2/3 during phagocytosis

We report herein that the anti-CD20 therapeutic antibody, rituximab, is rearranged into microclusters within the phagocytic synapse by macrophage Fcγ receptors (FcγR) during antibody-dependent cellular phagocytosis. These microclusters were observed to potently recruit Syk and to undergo rearrangements that were limited by the cytoskeleton of the target cell, with depolymerization of target-cell actin filaments leading to modest increases in phagocytic efficiency. Total internal reflection fluorescence analysis revealed that FcγR total phosphorylation, Syk phosphorylation, and Syk recruitment were enhanced when IgG-FcγR microclustering was enabled on fluid bilayers relative to immobile bilayers in a process that required Arp2/3. We conclude that on fluid surfaces, IgG-FcγR microclustering promotes signaling through Syk that is amplified by Arp2/3-driven actin rearrangements. Thus, the surface mobility of antigens bound by IgG shapes the signaling of FcγR with an unrecognized complexity beyond the zipper and trigger models of phagocytosis.

Tamoxifen Upregulates Collagenase Gene Expression in Human Dermal Fibroblasts.

Tamoxifen is a known inhibitor of fibroblast transforming growth factor beta biosynthesis and wound scar formation. Tamoxifen is also known to be an estrogen antagonist and protein kinase C (PKC) inhibitor. Cells treated with tamoxifen and other PKC/calmodulin inhibitors depolymerize their membrane focal adhesion complexes and cytoskeletal protein structures. These effects result in substrate detachment, cell shape rounding, and upregulation of collagenase synthesis and extracellular matrix degradation. The purpose of our study was to test the hypothesis that tamoxifen treatment of human foreskin fibroblasts results in alteration of cytoskeletal protein organization, cell detachment and rounding, and increased collagenase synthesis similar to known PKC/calmodulin inhibitors such as H-7. We characterized the effects of PKC/calmodulin inhibitors tamoxifen and H-7 on human dermal fibroblast morphology, cytoskeletal protein organization, and collagenase gene expression in monolayer culture and within collagen gels. We found that fibroblasts responded to tamoxifen by initiation of actin filament depolymerization followed by alteration from spindle to spheroidal shapes. This change in cell shape led to increased collagenase synthesis in cells treated with either tamoxifen or H-7 compared with controls. There was also a 23% increase of hydroxyproline release from tamoxifen-treated fibroblast-populated collagen matrices. Tamoxifen may reduce scarring by inhibiting fibroblast PKC/calmodulin activity, which down-regulates pro-fibrotic transforming growth factor beta signaling and upregulates collagenase production. These effects mimic those of the known PKC/calmodulin inhibitor H-7. Overall, these findings suggest that tamoxifen and its analogues are promising agents for clinical investigation as small molecule regulators of fibrosis and scarring disorders.

Drebrin Protects Assembled Actin from INF2-FFC-mediated Severing and Stabilizes Cell Protrusions

Highly specialized cells, such as neurons and podocytes, have arborized morphologies that serve their specific functions. Actin cytoskeleton and its associated proteins are responsible for the distinctive shapes of cells. The mechanism of their cytoskeleton regulation – contributing to cell shape maintenance – is yet to be fully clarified. Inverted formin 2 (INF2), one of the modulators of the cytoskeleton, is an atypical formin that can both polymerize and depolymerize actin filaments depending on its molar ratio to actin. Prior work has established that INF2 binds to the sides of actin filaments and severs them. Drebrin is another actin-binding protein that also binds filaments laterally and stabilizes them, but the interplay between drebrin and INF2 on actin filament stabilization is not well understood. Here, we have used biochemical assays, electron microscopy, and total internal reflection fluorescence microscopy imaging to show that drebrin protects actin filaments from severing by INF2 without inhibiting its polymerization activity. Notably, truncated drebrin – DrbA1-300 – is sufficient for this protection, though not as effective as the full-length protein. INF2 and drebrin are abundantly expressed in highly specialized cells and are crucial for the temporal regulation of their actin cytoskeleton, consistent with their involvement in peripheral neuropathy.

Depolymerization of actin filaments by Cucurbitacin I through binding G-actin.

Cucurbitacins have high economic value as they are a major source of food and have pharmacological properties. Cucurbitacin I (CuI) is a plant-derived natural tetracyclic triterpenoid compound that shows an anticancer effect via inhibiting the JAK2-STAT3 signaling pathway. The actin cytoskeleton is the most abundant protein in cells and regulates critical events through reorganization in cells. In this study, it is aimed at determining the direct effect of CuI on actin dynamics. The fluorescence profile of G-actin in the presence of CuI (1-200 nM) shifted to a higher temperature, suggesting that G-actin binds CuI and that G-actin-CuI is more thermally stable than the ligand-free form. CuI dose-dependently inhibited the polymerization of F-actin invitro and disrupted actin filaments in endothelial cells. Docking and MD simulations suggested that CuI binds to the binding site formed by residues I136, I175, D154, and A138 that are at the interface of monomers in F-actin. The migration ability of cells treated with CuI for 24 h was significantly lower than the control group (p < .001). This study reveals the molecular mechanisms of CuI in the regulation of actin dynamics by binding G-actin. More importantly, this study indicates a novel role of CuI as an actin-targeting drug by binding directly to G-actin and may contribute to the mode of action of CuI on anticancer activities.

Mass Spectrometry as a Quantitative Proteomic Analysis Tool for the Search for Temporal Lobe Epilepsy Biomarkers: A Systematic Review.

Temporal lobe epilepsy (TLE) is the most common form of epilepsy in adults. Tissue reorganization at the site of the epileptogenic focus is accompanied by changes in the expression patterns of protein molecules. The study of mRNA and its corresponding proteins is crucial for understanding the pathogenesis of the disease. Protein expression profiles do not always directly correlate with the levels of their transcripts; therefore, it is protein profiling that is no less important for understanding the molecular mechanisms and biological processes of TLE. The study and annotation of proteins that are statistically significantly different in patients with TLE is an approach to search for biomarkers of this disease, various stages of its development, as well as a method for searching for specific targets for the development of a further therapeutic strategy. When writing a systematic review, the following aggregators of scientific journals were used: MDPI, PubMed, ScienceDirect, Springer, and Web of Science. Scientific articles were searched using the following keywords: "proteomic", "mass-spectrometry", "protein expression", "temporal lobe epilepsy", and "biomarkers". Publications from 2003 to the present have been analyzed. Studies of brain tissues, experimental models of epilepsy, as well as biological fluids, were analyzed. For each of the groups, aberrantly expressed proteins found in various studies were isolated. Most of the studies omitted important characteristics of the studied patients, such as: duration of illness, type and response to therapy, gender, etc. Proteins that overlap across different tissue types and different studies have been highlighted: DPYSL, SYT1, STMN1, APOE, NME1, and others. The most common biological processes for them were the positive regulation of neurofibrillary tangle assembly, the regulation of amyloid fibril formation, lipoprotein catabolic process, the positive regulation of vesicle fusion, the positive regulation of oxidative stress-induced intrinsic apoptotic signaling pathway, removal of superoxide radicals, axon extension, and the regulation of actin filament depolymerization. MS-based proteomic profiling for a relevant study must accept a number of limitations, the most important of which is the need to compare different types of neurological and, in particular, epileptic disorders. Such a criterion could increase the specificity of the search work and, in the future, lead to the discovery of biomarkers for a particular disease.

DENND2B activates Rab35 at the intercellular bridge, regulating cytokinetic abscission and tetraploidy

Cytokinesis relies on membrane trafficking pathways regulated by Rabs and guanine nucleotide exchange factors (GEFs). During cytokinesis, the intercellular cytokinetic bridge (ICB) connecting daughter cells undergoes abscission, which requires actin depolymerization. Rab35 recruits MICAL1 to oxidize and depolymerize actin filaments. We show that DENND2B, a protein linked to cancer and congenital disorders, functions as a Rab35 GEF, recruiting and activating Rab35 at the ICB. DENND2B's N-terminal region also interacts with an active form of Rab35, suggesting that DENND2B is both a Rab35 GEF and effector. Knockdown of DENND2B delays abscission, leading to multinucleated cells and filamentous actin (F-actin) accumulation at the ICB, impairing recruitment of ESCRT-III at the abscission site. Additionally, F-actin accumulation triggers the formation of a chromatin bridge, activating the NoCut/abscission checkpoint, and DENND2B knockdown activates Aurora B kinase, a hallmark of checkpoint activation. Thus, our study identifies DENND2B as a crucial player in cytokinetic abscission.

Mechanical stretch facilitates tenomodulin expression to induce tenocyte migration via MAPK signaling pathway

Tenomodulin (Tnmd) is a type II transmembrane glycoprotein that regulates tendon development and maturation. Our previous study indicated that mechanical stretch could induce Tnmd expression to promote tenocyte migration, associated with reinforcement of fibrous actin (F-actin) stress fibers and chromatin decondensation. However, the detailed molecular mechanisms of this processes are far from clear. Activation of mitogen-activated protein kinase (MAPK) signaling occurs in response to various extracellular stimuli and controls a large number of fundamental cellular processes. The present study we investigated the influence of MAPK signaling on mechanical stretch-induced Tnmd expression and its action way. Expression and activities of extracellular signal-related kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38 MAPK (p38) were determined by Western blot. Cell migration was detected by Transwell assay. Immunofluorescence staining was used to detect F-actin stress fibers. Nuclear chromatin decondensation was detected by in situ DNaseI sensitivity assay. It was found that mechanical stretch promoted Tnmd expression by activating ERK1/2, JNK and p38 signaling. The inhibition of the ERK1/2, JNK or p38 repressed mechanical stretch-promoted tenocyte migration and mechanical stretch-induced reinforcement of F-actin stress fibers. However, only ERK1/2 and p38 inhibitor could repress mechanical stretch-induced chromatin decondensation, and the JNK inhibitor had no significant effect. Moreover, latrunculin (Lat A), the most widely used reagent to depolymerize actin filaments, could inhibit the stretch-induced chromatin decondensation. Taken together, our findings elucidated a molecular pathway by which a mechanical signal is transduced via activation of MAPK signaling to influence reinforcement of F-actin stress fibers and chromatin decondensation, which could further lead Tnmd expression to promote tenocyte migration.

Differential expression of Mical2 and its activator (Sema3a) in Alzheimer’s disease mouse model

AbstractBackgroundMicals are actin regulatory enzymes which can enhance depolymerization of actin filaments (F‐Actin). Activated Micals catalyze the selective oxidation of methionine residues, Met44 and Met47 within the D‐loop of F‐actin. The oxidation of methionine residues disrupts the interaction between G‐actin monomers, thus facilitating F‐actin depolymerization. Dendritic spines are enriched with filamentous actin (F‐actin) where it regulates the formation, elimination and structural plasticity of spines. Early stage of AD is associated with loss of dendritic spines and F‐actin from synapses, leading to memory deficits. However, the potential mechanism involved in the loss of F‐actin remains largely unknown. Therefore, we aimed to elucidate the role of Micals and semaphorins in actin depolymerization at the synapse of AD mouse model.MethodsTotal RNA was isolated from the cortex, hippocampus and entorhinal cortex of adolescent, young, and middle aged wildtype and APP/PS1 mice as well as from the synaptosomes of respective age groups. The cDNA is synthesized and qRT‐PCR is performed using SYBR green reagents with specific primers to Micals and Semaphorins. Data was analyzed using two tailed non parametric test with a p value of &lt;0.05 signifies the result.ResultsThe mRNA expression levels of Micals and Semaphorins in entorhinal cortex, hippocampus, and cortex of wildtype and APP/PS1 as well as in the synaptosomes of age matched animals were measured by qRT‐PCR. We found that Semaphorin3a showed differential mRNA expression levels in entorhinal cortex of adolescence and young adults of APP/PS1 mice in comparison to wildtype littermate controls. Further, we detected that the synapse specific mRNA levels of Mical 2 and Semaphorin3a are significantly decreased in adolescent APP/PS1 mice in comparison to WT mice. However, we did not detect any significant alteration in the mRNA expression levels of Mical1, Mical3 and Semaphorin3f in APP/PS1 mice at different age groups in comparison to WT littermate controls.ConclusionAltered expression levels of actin regulatory proteins like Micals and Semaphorins at the dendritic spines could play critical roles in Aβ mediated F‐actin depolymerization in Alzheimer's disease pathology.

LRRK2 decreases microglial actin dynamics by filamentous actin depolymerization and Rac1 inhibition

ABSTRACT An active actin dynamic is a crucial feature of brain microglia. Here we report that LRRK2, a primary familial Parkinson’s disease-associated gene, negatively regulates microglia’s actin dynamics. LRRK2 depolymerized filamentous actin (F-actin) by directly binding to it or inhibiting microglia’s Rac-PAK signaling. LRRK2 knockdown resulted in a reduced ruffle and enhanced lamellipodia formation of ADP-activated microglia, altering the microglia’s physiological activity to vigorous migration toward damaged cells. These results suggest that LRRK2 is a negative regulator for the controlled actin dynamics in microglia.

Aphelenchoides besseyi Ab-FAR-1 Interacts with Arabidopsis thaliana AtADF3 to Interfere with Actin Cytoskeleton, and Promotes Nematode Parasitism and Pathogenicity.

Fatty acid and retinol binding proteins (FAR) are unique proteins found in nematodes and are considered potential targets for controlling these parasites. However, their functions in nematode parasitism and pathogenicity and interaction with hosts are still unclear. In this study, we investigated the specific roles of rice white tip nematodes (RWTNs), Aphelenchoides besseyi, and a protein, Ab-FAR-1, to elucidate the parasitic and pathogenic processes of nematodes. The results showed that the expression level of Ab-far-1 was significantly up-regulated after A. besseyi infection of the plant. The immunofluorescence and subcellular localisation showed that Ab-FAR-1 was secreted into plant tissues mainly through the body wall of nematodes and might act in the nucleus and cytoplasm of plant cells. The pathogenicity of RWTNs was enhanced in Arabidopsis thaliana overexpressing Ab-FAR-1 and inhibited in Ab-far-1 RNAi A. thaliana. Yeast two-hybrid, Co-IP, BiFC, and nematode inoculation experiments showed that Ab-FAR-1 could interact with the A. thaliana actin-depolymerizing factor protein AtADF3, and the A. thaliana adf3 mutant was more susceptible to nematodes. An in vitro actin filament depolymerisation assay demonstrated that Ab-FAR-1 could inhibit AtADF3-mediated depolymerisation of actin filaments, and the turnover process of cellular actin filaments was also affected in A. thaliana overexpressing Ab-FAR-1. In addition, flg22-mediated host defence responses were suppressed in A. thaliana overexpressing Ab-FAR-1 and adf3 mutants. Therefore, this study confirmed that RWTNs can affect the turnover of actin filament remodelling mediated by AtADF3 through Ab-FAR-1 secretion and thus inhibit plant PAMP-triggered immunity (PTI), promoting the parasitism and pathogenicity of nematodes.

F-actin dynamics in midgut cells enables virus persistence in vector insects.

Hemipteran insects that transmit plant viruses in a persistent circulative manner acquire, retain and transmit viruses for their entire life. The mechanism enabling this persistence has remained unclear for many years. Here, we determined how wheat dwarf virus (WDV) persists in its leafhopper vector Psammotettix alienus. We found that WDV caused the up‐regulation of actin‐depolymerizing factor (ADF) at the mRNA and protein levels in the midgut cells of leafhoppers after experiencing a WDV acquisition access period (AAP) of 6, 12 or 24 h. Experimental inhibition of F‐actin depolymerization by jasplakinolide and dsRNA injection led to lower virus accumulation levels and transmission efficiencies, suggesting that depolymerization of F‐actin regulated by ADF is essential for WDV invasion of midgut cells. Exogenous viral capsid protein (CP) inhibited ADF depolymerization of actin filaments in vitro and in Spodoptera frugiperda 9 (Sf9) cells because the CP competed with actin to bind ADF and then blocked actin filament disassembly. Interestingly, virions colocalized with ADF after a 24‐h AAP, just as actin polymerization occurred, indicating that the binding of CP with ADF affects the ability of ADF to depolymerize F‐actin, inhibiting WDV entry. Similarly, the luteovirus barley yellow dwarf virus also induced F‐actin depolymerization and then polymerization in the gut cells of its vector Schizaphis graminum. Thus, F‐actin dynamics are altered by nonpropagative viruses in midgut cells to enable virus persistence in vector insects.

Reversible Deformation of Artificial Cell Colonies Triggered by Actin Polymerization for Muscle Behavior Mimicry.

The use of artificial cells to mimic living tissues is beneficial for understanding the mechanism of interaction among cells. Artificial cells hold immense potential in the field of tissue engineering. Self-powered artificial cells capable of reversible deformation are developed by encapsulating living mitochondria, actins, and methylcellulose. Upon addition of pyruvate molecules, the mitochondria produce adenosine triphosphate (ATP), which acts as an energy source to trigger actin polymerization. The reversible deformation of artificial cells occurs with a spindle shape resulting from the polymerization of actins to form filaments adjacent to the lipid bilayer that subsequently returns to a spherical shape resulting from the depolymerization of actin filaments upon laser irradiation. The linear colonies composed of these artificial cells exhibit collective contraction and relaxation to mimic muscle tissues. At maximum contraction, the long axis of each giant unilamellar vesicle (GUV) is parallel to each other. All the colonies are synchronized in the contraction phase. The deformation of each GUV in the colonies is influenced by its adjacent GUVs. The muscle-like artificial cell colonies described here pave the way to develop sustainably self-powered artificial tissues.

LIM Kinases, Promising but Reluctant Therapeutic Targets: Chemistry and Preclinical Validation In Vivo.

LIM Kinases are important actors in the regulation of cytoskeleton dynamics by controlling microtubule and actin filament turnover. The signaling pathways involving LIM kinases for actin filament remodeling are well established. They are downstream effectors of small G proteins of the Rho-GTPases family and have become promising targets for the treatment of several major diseases because of their position at the lower end of these signaling cascades. Cofilin, which depolymerizes actin filaments, is the best-known substrate of these enzymes. The phosphorylation of cofilin to its inactive form by LIM kinases avoids actin filament depolymerization. The balance between phosphorylated and non-phosphorylated cofilin is thought to play an important role in tumor cell invasion and metastasis. Since 2006, many small molecules have been developed for LIMK inhibition, and in this review article, we will discuss the structure–activity relationships of the few inhibitor families that have been tested in vivo on different pathological models.

Spindle motility skews division site determination during asymmetric cell division in Physcomitrella.

Asymmetric cell division (ACD) underlies the development of multicellular organisms. In animal ACD, the cell division site is determined by active spindle-positioning mechanisms. In contrast, it is considered that the division site in plants is determined prior to mitosis by the microtubule-actin belt known as the preprophase band (PPB) and that the localization of the mitotic spindle is typically static and does not govern the division plane. However, in some plant species, ACD occurs in the absence of PPB. Here, we isolate a hypomorphic mutant of the conserved microtubule-associated protein TPX2 in the moss Physcomitrium patens (Physcomitrella) and observe spindle motility during PPB-independent cell division. This defect compromises the position of the division site and produces inverted daughter cell sizes in the first ACD of gametophore (leafy shoot) development. The phenotype is rescued by restoring endogenous TPX2 function and, unexpectedly, by depolymerizing actin filaments. Thus, we identify an active spindle-positioning mechanism that, reminiscent of acentrosomal ACD in animals, involves microtubules and actin filaments, and sets the division site in plants.

Examination of the Interplay between Membrane Tension and the Tight Junction Protein, ZO‐1

Zonula occluden‐1 (ZO‐1) has been implicated as a major regulator of tight junction formation, functioning as a scaffolding protein and mediator between the junction and the actomyosin cytoskeleton. MDCK II cells that have ZO‐1 knocked‐down (ZO‐1 KD) or knocked‐out (ZO‐1 KO) have observable phenotypic changes, including increased apical actin accumulation, increased cell height, and linearized junctions. The apical actin accumulation, resulting from the dysregulation of myosin II, is suspected to cause an increase in membrane tension seen in the ZO‐1 KO, which may explain the phenotypic changes. The function of ZO‐1 as a regulator of myosin II was confirmed by exposing the ZO‐1 KO to blebbistatin, a myosin II inhibitor, which restored the cell height to that of wild‐type MDCK II cells (WT) and the curvature of the junctions. Flipper‐TR, which is a live cell fluorescent membrane tension probe, along with fluorescence‐lifetime imaging microscopy (FLIM) analysis, are being used to test the membrane tension of the ZO‐1 KO cells compared to WT cells. The Flipper‐TR probe works by inserting itself into a lipid membrane where it undergoes conformational changes induced by membrane tension. The relationship between membrane tension and actin was examined using latrunculin A (LatA), which causes the depolymerization of actin filaments. When both the ZO‐1 KO and WT cells were incubated with LatA, the membrane tension of both decreased to a similar extent. The preliminary results of this testing have shown an increased membrane tension in the ZO‐1 KO cells. To measure the response of ZO‐1 KO cells to increased tension, the expression of tension‐related genes is being measured using quantitative real‐time polymerase chain reaction (qRT‐PCR). These tension‐related genes include the ERM (ezrin, radixin, and moesin) proteins, with a primary interest in ezrin given its known activity as a major regulator of membrane tension in epithelial cells. This study aims to understand the mechanism between ZO‐1 loss and regulation of membrane tension.

Induction of Filopodia During Cytomegalovirus Entry Into Human Iris Stromal Cells.

Many viruses exploit thin projections of filopodia for cell entry and cell-to-cell spread. Using primary cultures of human iris stromal (HIS) cells derived from human eye donors, we report a significant increase in filopodia formation during human cytomegalovirus (HCMV) infection. Using confocal microscopy, we observed a large number of virions being frequently associated along the filopodia prior to cell infection. Depolymerization of actin filaments resulted in a significant inhibition of HCMV entry into HIS cell. Our results further revealed that the transient expression of HCMV envelope glycoprotein B (gB) triggers the induction of the filopodial system. Since gB is known to bind the diverse chains of heparan sulfate (HS), a comparative study was performed to evaluate the gB-mediated filopodial induction in cells expressing either wild-type HS and/or 3-O sulfated HS (3-OS HS). We found that cells co-expressing HCMV gB together with the 3-O sulfotranseferase-3 (3-OST-3) enzyme had a much higher and robust filopodia induction compared to cells co-expressing gB with wild-type HS. The above results were further verified by pre-treating HIS cells with anti-3-OS HS (G2) peptide and/or heparinase-I before challenging with HCMV infection, which resulted in a significant loss in the filopodial counts as well as decreased viral infectivity. Taken together, our findings highlight that HCMV entry into HIS cells actively modulates the actin cytoskeleton via coordinated actions possibly between gB and the 3-OS HS receptor to influence viral infectivity.

Functional non‐equivalence of pollenADFisovariants in Arabidopsis

ADF/cofilin is a central regulator of actin dynamics. We previously demonstrated that two closely related Arabidopsis class IIa ADF isovariants, ADF7 and ADF10, are involved in the enhancement of actin turnover in pollen, but whether they have distinct functions remains unknown. Here, we further demonstrate that they exhibit distinct functions in regulating actin turnover both in vitro and in vivo. We found that ADF7 binds to ADP-G-actin with lower affinity, and severs and depolymerizes actin filaments less efficiently in vitro than ADF10. Accordingly, in pollen grains, ADF7 more extensively decorates actin filaments and is less freely distributed in the cytoplasm compared to ADF10. We further demonstrate that ADF7 and ADF10 show distinct intracellular localizations during pollen germination, and they have non-equivalent functions in promoting actin turnover in pollen. We thus propose that cooperation and labor division of ADF7 and ADF10 enable pollen cells to achieve exquisite control of the turnover of different actin structures to meet different cellular needs.

Microtubules play a crucial role in regulating actin organization and cell initiation in cotton fibers.

Dynamic organization of actin and microtubule cytoskeletons directs a distinct expansion behavior of cotton fiber initiation from cell elongation. Cotton fibers are highly elongated single cells derived from the ovule epidermis. Although actin and microtubule (MT) cytoskeletons have been implicated in cell elongation and secondary wall deposition, their roles in fiber initiation is poorly understood. Here, we used fluorescent probes and pharmacological approaches to study the roles of these cytoskeletal components during cotton fiber initiation. Both cytoskeletons align along the growth axis in initiating fibers. The dorsal view of ovules shows that unlike the fine actin filaments (AFs) in nonfiber cells, the AFs in fiber cells are dense and bundled. MTs are randomized in fiber cells and well-ordered in nonfiber cells. The pharmacological experiments revealed that the depolymerization of AFs and MTs assisted fiber initiation. Both AF stabilization and depolymerization inhibited fiber elongation. In contrast, the proper depolymerization of MTs promoted cell elongation, although the MT-stabilizing drug consistently resulted in a negative effect. Notably, we found that the organization of AFs was correlated with MT dynamics. Stabilizing the MTs by taxol treatment promoted the formation of AF bundles (in fiber initials) and transversely aligned AFs (in elongating fibers), whereas depolymerizing the MTs by oryzalin treatment promoted the fragmentation of AFs. Collectively, our data indicates that MTs plays a crucial role in regulating AF organization and early development of cotton fibers.