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The plant cytoskeleton persistently undergoes remodeling to achieve its roles in supporting cell division, differentiation, cell expansion and organelle transport. However, the links between cell metabolism and cytoskeletal networks, particularly how the proteinaceous components execute such processes remain poorly understood. We investigated the cytoskeletal proteome landscape of rice to gain better understanding of such events. Proteins were extracted from highly enriched cytoskeletal fraction of four-week-old rice seedlings, and the purity of the fraction was stringently monitored. A total of 2577 non-redundant proteins were identified using both gel-based and gel-free approaches, which constitutes the most comprehensive dataset, thus far, for plant cytoskeleton. The data set includes both microtubule and microfilament-associated proteins and their binding proteins comprising hypothetical as well as novel cytoskeletal proteins. Further, various in-silico analyses were performed, and the proteins were functionally classified on the basis of their gene ontology. The catalogued proteins were validated through their sequence analysis. Extensive comparative analysis of our dataset with the non-redundant set of cytoskeletal proteins across plant species affirms unique as well as overlapping candidates. Together, these findings unveil new insights of how cytoskeletons undergo dynamic remodeling in rice to drive seedling development processes in rapidly changing in planta environment.

Zearalenone (ZEN) is a worldwide fusarotoxin that poses a threat to the consumer due to its chronic toxicity. Herein we examined the effects of ZEN on adult mouse testis, focusing on oxidative stress, biochemical and morphological parameters. In addition, since cytoskeletal remodeling is a key event for the production of good quality gametes, the expression and localization of two proteins, Dishevelled-associated activator of morphogenesis 1 (DAAM1) and Prolyl endopeptidase (PREP), involved in cytoskeletal dynamics during spermatogenesis were evaluated. To ameliorate the testicular dysfunction induced by ZEN we tested the eventual protective effects of lactic bacteria Lactobacillus plantarum MON03 (LP) on its reprotoxicity. Adult male mice were then treated daily for 2 wks by oral gavage with ZEN and/or LP. The results confirmed that ZEN altered sperm parameters, generated oxidative stress and provoked structural alteration, evidenced by the increased number of abnormal seminiferous tubules and of apoptotic cells, particularly Leydig cells. Interestingly, at molecular level we evaluated, for the first time, the ability of ZEN to alter DAAM1 and PREP protein level and localization. Moreover, the co-treatment with LP, thanks to its capacity to reduce ZEN bioavailability in the gastrointestinal tract, ameliorated all the considered parameters. These results suggest the use of this probiotic as food supplement to prevent/counteract ZEN-induced reprotoxicity.

MicroRNAs (miRNAs) play an essential role in the regulation of a number of physiological functions. miR-133a and other muscular miRs (myomiRs) play a key role in muscle cell growth and in some type of cancers. Here, we show that miR133a is upregulated in individuals that undertake physical exercise. We used a skeletal muscle differentiation model to dissect miR-133a’s role and to identify new targets, identifying Tropomyosin-4 (TPM4). This protein is expressed during muscle differentiation, but importantly it is an essential component of microfilament cytoskeleton and stress fibres formation. The microfilament scaffold remodelling is an essential step in cell transformation and tumour progression. Using the muscle system, we obtained valuable information about the microfilament proteins, and the knowledge on these molecular players can be transferred to the cytoskeleton rearrangement observed in cancer cells. Further investigations showed a role of TPM4 in cancer physiology, specifically, we found that miR-133a downregulation leads to TPM4 upregulation in colon carcinoma (CRC), and this correlates with a lower patient survival. At molecular level, we demonstrated in myocyte differentiation that TPM4 is positively regulated by the TA isoform of the p63 transcription factor. In muscles, miR-133a generates a myogenic stimulus, reducing the differentiation by downregulating TPM4. In this system, miR-133a counteracts the differentiative TAp63 activity. Interestingly, in CRC cell lines and in patient biopsies, miR-133a is able to regulate TPM4 activity, while TAp63 is not active. The downregulation of the miR leads to TPM4 overexpression, this modifies the architecture of the cell cytoskeleton contributing to increase the invasiveness of the tumour and associating with a poor prognosis. These results add data to the interesting question about the link between physical activity, muscle physiology and protection against colorectal cancer. The two phenomena have in common the cytoskeleton remodelling, due to the TPM4 activity, that is involved in stress fibres formation.

Human skin fibroblasts (HSFs) approximate the multidirectional differentiation potential of mesenchymal stem cells, so they are often used in differentiation, cell cultures, and injury repair. They are an important seed source in the field of bone tissue engineering. However, there are a few studies describing the mechanism of osteogenic differentiation of HSFs. Here, osteogenic induction medium was used to induce fibroblasts to differentiate into osteoblasts, and the role of the mechanical sensitive element PDLIM5 in microfilament-mediated osteogenic differentiation of human fibroblasts was evaluated. The depolymerization of microfilaments inhibited the expression of osteogenesis-related proteins and alkaline phosphatase activity of HSFs, while the polymerization of microfilaments enhanced the osteogenic differentiation of HSFs. The evaluation of potential protein molecules affecting changes in microfilaments showed that during the osteogenic differentiation of HSFs, the expression of PDLIM5 increased with increasing induction time, and decreased under the state of microfilament depolymerization. Lentivirus-mediated PDLIM5 knockdown by shRNA weakened the osteogenic differentiation ability of HSFs and inhibited the expression and morphological changes of microfilament protein. The inhibitory effect of knocking down PDLIM5 on HSF osteogenic differentiation was reversed by a microfilament stabilizer. Taken together, these data suggest that PDLIM5 can mediate the osteogenic differentiation of fibroblasts by affecting the formation and polymerization of microfilaments.

Actin is a major cytoskeletal protein that plays crucial roles in a number of biological events involving force generation and shape changes. Actin monomers are polymerized into actin filaments, which serve as a core of the actin cytoskeleton together with many associated proteins. Although purified actin can be spontaneously polymerized under physiological conditions in test tubes, assembly and disassembly of actin are spatially and temporally controlled within cells. For example, concerted directional assembly of actin filaments can push membranes and organelles, whereas disassembly of actin filaments contributes to cytoskeletal remodeling and recycling of disassembled actin monomers for a new round of actin polymerization. Therefore, coordinated regulation of actin assembly and disassembly is often required to achieve normal cellular behaviors. Particularly, actin filament disassembly is a challenging task in the cytoplasm. Once actin is polymerized, slow spontaneous dissociation of actin subunits from filaments limits the rate of overall actin turnover. In addition, the cytoplasm generally contains high concentrations of actin monomers that can increase net actin assembly. One of the factors that promote actin filament disassembly is the actin depolymerizing factor (ADF)/cofilin family of proteins, which is expressed in various cell types across eukaryotes and involved in cellular processes that require dynamic rearrangement of the actin cytoskeleton, such as cell migration, cytokinesis, and morphogenesis (1, 2). ADF/cofilin (hereafter referred to as cofilin) promotes actin depolymerization and enhances actin turnover (3⇓–5). Cofilin binds to the side of actin filaments at a 1:1 (cofilin:actin subunit) molar ratio in a cooperative manner such that clusters of cofilin-decorated regions are generated. Then, filament severing occurs frequently at or near boundaries between cofilin-decorated and bare regions on the filament (6, 7). Therefore, cofilin severs actin filaments most efficiently when cofilin binds to filaments at low densities (8). However, the mechanism of … [↵][1]1Email: sono{at}emory.edu. [1]: #xref-corresp-1-1

Background The combination of a microtubule inhibitor (eribulin) with a nucleoside analog (gemcitabine) may synergistically induce tumor cell death, particularly in triple negative breast cancer (TNBC) characterized by high cell proliferation, aggressive behavior, and chemo-resistance. Patients and methods This is an open-label, multicenter phase II study evaluating the combination of eribulin (0.88 mg/m2) plus gemcitabine (1000 mg/m2) on days 1 and 8 of a 21-day cycle as either first- or second-line treatment of locally advanced or metastatic TNBC. The primary endpoint was the objective response for evaluable patients. A prospective, molecular correlative study was carried out to assess the role of germinal BRCA pathogenic variants and single nucleotide polymorphisms (SNPs) in predicting efficacy and toxicity of the combination regimen. Results From July 2013 to September 2016, 83 evaluable patients were enrolled. They received a median number of six cycles of treatment. An overall response rate (ORR) of 37.3% (31 patients) was observed, with a complete response rate of 2.4% and a partial response rate of 34.9%; the clinical benefit rate was 48.8%. With a median follow-up of 28.8 months, the median response duration was 6.6 months, the median progression-free survival (PFS) was 5.1 months, and the median overall survival (OS) was 14.5 months. The most common grade 3-4 adverse events were aminotransferase elevation (in 25% of the patients) and neutropenia (in 23.8%). Women with BRCA1/2 pathogenic variants were associated with worse ORR, PFS, and OS than BRCA1/2 wild-type carriers. CYP3A4 and FGD4 SNPs were associated with increased risk of liver toxicity. Three different SNPs in CDA∗2, RRM1, and CYP2C8 genes were significantly associated with poorer OS. Conclusions The combination of eribulin and gemcitabine showed promising activity and a moderate toxicity profile in metastatic TNBC. BRCA status and pharmacogenetics tests may help identify patients with high probability of response with negligible toxicity. EudraCT number 2012-003505-10., Highlights • Eribulin plus gemcitabine showed a remarkable best ORR of 37.3% and a clinical benefit rate of 48.8%. • The most common grade 3/4 toxicities were liver toxicity and neutropenia without febrile neutropenia. • The study regimen partially lost its efficacy in patients harboring BRCA1/2 pathogenic variants. • SNPs in CYP3A4 and FGD4 genes were associated with increased risk of liver toxicity. • Three different SNPs in CDA∗2, RRM1, and CYP2C8 genes were significantly associated with poorer OS.

Constriction of the cytokinetic ring, a circular structure of actin filaments, is an essential step during cell division. Mechanical forces driving the constriction are attributed to myosin motor proteins, which slide actin filaments along each other. However, in multiple organisms, ring constriction has been reported to be myosin independent. How actin rings constrict in the absence of motor activity remains unclear. Here, we demonstrate that anillin, a non­motor actin crosslinker, indispensable during cytokinesis, autonomously propels the contractility of actin bundles. Anillin generates contractile forces of tens of pico-Newtons to maximise the lengths of overlaps between bundled actin filaments. The contractility is enhanced by actin disassembly. When multiple actin filaments are arranged into a ring, this contractility leads to ring constriction. Our results indicate that passive actin crosslinkers can substitute for the activity of molecular motors to generate contractile forces in a variety of actin networks, including the cytokinetic ring., Cytokinetic ring constriction during cell division requires actin but curiously is independent of myosin in many organisms. Here, the authors show that anillin, a protein enriched in the contractile ring, is a non-motor actin crosslinker that generates contractile force in lieu of a molecular motor.

The mechanisms that modulate the kinetics of muscle relaxation are critically important for muscle function. A prime example of the impact of impaired relaxation kinetics is nemaline myopathy caused by mutations in KBTBD13 (NEM6). In addition to weakness, NEM6 patients have slow muscle relaxation, compromising contractility and daily life activities. The role of KBTBD13 in muscle is unknown, and the pathomechanism underlying NEM6 is undetermined. A combination of transcranial magnetic stimulation–induced muscle relaxation, muscle fiber– and sarcomere-contractility assays, low-angle x-ray diffraction, and superresolution microscopy revealed that the impaired muscle-relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament, a sarcomeric microstructure. Using homology modeling and binding and contractility assays with recombinant KBTBD13, Kbtbd13-knockout and Kbtbd13(R408C)-knockin mouse models, and a GFP-labeled Kbtbd13-transgenic zebrafish model, we discovered that KBTBD13 binds to actin — a major constituent of the thin filament — and that mutations in KBTBD13 cause structural changes impairing muscle-relaxation kinetics. We propose that this actin-based impaired relaxation is central to NEM6 pathology.

By using an approach that allows computing the free energy in high-dimensional spaces together with a clustering technique capable of identifying kinetic attractors stabilized by conformational disorder, we analyze a molecular dynamics trajectory of the Villin headpiece from Lindorff-Larsen, K.; et al. How fast-folding proteins fold. Science 2011, 334, 517-520. We compute its free-energy landscape in the space of all its Cα carbons. This landscape has the shape of a 12-dimensional funnel with the free energy decreasing monotonically as a function of the native contacts. There are no significant folding barriers. The funnel can be partitioned in five regions, three mainly folded and two unfolded, which behave as Markov states. The slowest relaxation time among these states corresponds to the folding transition. The second slowest time is only twice smaller and corresponds to a transition within the unfolded state. This indicates that the unfolded part of the funnel has a nontrivial shape, which induces a sizable kinetic barrier between disordered states.

Molecule interacting with CasL 2 (MICAL2), a cytoskeleton dynamics regulator, are strongly expressed in several human cancer types, especially at the invasive front, in metastasizing cancer cells and in the neo-angiogenic vasculature. Although a plethora of data exist and stress a growing relevance of MICAL2 to human cancer, it is worth noting that only one small-molecule inhibitor, named CCG-1423 (1), is known to date. Herein, with the aim to develop novel MICAL2 inhibitors, starting from CCG-1423 (1), a small library of new compounds was synthetized and biologically evaluated on human dermal microvascular endothelial cells (HMEC-1) and on renal cell adenocarcinoma (786-O) cells. Among the novel compounds, 10 and 7 gave interesting results in terms of reduction in cell proliferation and/or motility, whereas no effects were observed in MICAL2-knocked down cells. Aside from the interesting biological activities, this work provides the first structure–activity relationships (SARs) of CCG-1423 (1), thus providing precious information for the discovery of new MICAL2 inhibitors.

In this issue of Blood, Thurner et al identified that two-thirds of patients with primary central nervous system lymphoma (PCNSL) have B-cell receptors (BCRs) specific for 2 autoantigens, SAMD14 and neurabin-1. These antigens are predominantly expressed in the central nervous system (CNS), induced strong BCR pathway activation, and autorecognition of them is therefore an attractive explanation for the CNS tropism of the disease.1

A tyrosine kinase connects tension with strength of junctions between three adjacent cells

Germ cells in most animals are connected by intercellular bridges, actin-based rings that form stable cytoplasmic connections between cells promoting communication and coordination [1]. Moreover, these connections are required for fertility [1, 2]. Intercellular bridges are proposed to arise from stabilization of the cytokinetic ring during incomplete cytokinesis [1]. Paradoxically, proteins that promote closure of cytokinetic rings are enriched on stably open intercellular bridges [1, 3, 4]. Given this inconsistency, the mechanism of intercellular bridge stabilization is unclear. Here, we used the C. elegans germline as a model for identifying molecular mechanisms regulating intercellular bridges. We report that bridges are actually highly dynamic, changing size at precise times during germ cell development. We focused on the regulation of bridge stability by anillins, key regulators of cytokinetic rings and cytoplasmic bridges [1, 4-7]. We identified GCK-1, a conserved serine/threonine kinase [8], as a putative novel anillin interactor. GCK-1 works together with CCM-3, a known binding partner [9], to promote intercellular bridge stability and limit localization of both canonical anillin and non-muscle myosin II (NMM-II) to intercellular bridges. Additionally, we found that a shorter anillin, known to stabilize bridges [4, 7], also regulates NMM-II levels at bridges. Consistent with these results, negative regulators of NMM-II stabilize intercellular bridges in the Drosophila egg chamber [10, 11]. Together with our findings, this suggests that tuning of myosin levels is a conserved mechanism for the stabilization of intercellular bridges that can occur by diverse molecular mechanisms.

Cardiac myofilaments act as the central contractile apparatus of heart muscle cells. Covalent modification of constituent proteins through phosphorylation is a rapid and powerful mechanism to control myofilament function, and is increasingly seen as a mechanism of disease. While the relationship between protein kinases and cardiac myofilaments has been widely examined, the impact of protein dephosphorylation by protein phosphatases is poorly understood. This review outlines the mechanisms by which the mostly widely expressed protein phosphatases in cardiac myocytes regulate myofilament function, and the emerging role of myofilament-associated protein phosphatases in heart failure. The importance of regulatory subunits and subcellular compartmentalization in determining the functional impact of protein phosphatases on myofilament and myocardial function is also discussed, as are discrepancies about the roles of protein phosphatases in regulating myofilament function. The potential for targeting these molecular messengers in the treatment of heart failure is discussed as a key future direction.

Study of Drosophila oogenesis reveals that the nuclear localization of actin is controlled by both development and Fascin. Fascin regulates both endogenous nuclear actin and ectopic nuclear actin rod formation by controlling Cofilin., Drosophila oogenesis provides a developmental system with which to study nuclear actin. During Stages 5–9, nuclear actin levels are high in the oocyte and exhibit variation within the nurse cells. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged Actin results in nuclear actin rod formation. These findings indicate that nuclear actin must be tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, whereas overexpression of Fascin increases, the frequency of nurse cells with high levels of nuclear actin, but neither alters the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate nuclear actin. Evidence indicates that Fascin positively regulates nuclear actin through Cofilin. Loss of Fascin results in decreased nuclear Cofilin. In addition, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high nuclear actin levels. These findings are likely applicable beyond Drosophila follicle development, as the localization and functions of Fascin and the mechanisms regulating nuclear actin are widely conserved.

The Coronin family is one of the WD-repeat domain containing families that are diverse in both of their structures and functions. The first coronin was identified in the cytoskeleton composition of Dictyostelium discoideum, which was discovered to regulate the actin functions. So far, 723 coronins have been identified throughout the eukaryotic kingdom by bioinformatics analysis in 358 species. In mammals, 7 coronins have been identified to date, which are named through Coronin 1 to Coronin 7; all of these isoforms contain two structurally conservational region: a 7-bladed β-propeller scaffold in N-terminal and a C-terminal variable coiled coil domain. Although some studies were showing that mammalian coronins have regulated the actin dynamics, recently many other functions such as calcium signaling regulation, cAMP signaling regulation, have been also reported beyond the actin modulation. Furthermore, many diseases have been found to be extensively associated with the abnormal expression of coronins, such as auto-immunity, bacterial and virus infection, neuronal behavior disorder and cancer. In this review, we would like to systematically discuss the recent progresses of mammalian coronins and associated diseases, as well as possible underlying molecular mechanisms.

Furrow ingression is asymmetric in cytokinesis in the Caenorhabditis elegans zygote. A combination of quantitative high-resolution live-cell microscopy and theoretical modeling revealed a mechanistic basis for asymmetry: feedback among membrane curvature, cytoskeletal alignment, and contractility. The model also suggests that asymmetry promotes energy efficiency., During cytokinesis, the cell undergoes a dramatic shape change as it divides into two daughter cells. Cell shape changes in cytokinesis are driven by a cortical ring rich in actin filaments and nonmuscle myosin II. The ring closes via actomyosin contraction coupled with actin depolymerization. Of interest, ring closure and hence the furrow ingression are nonconcentric (asymmetric) within the division plane across Metazoa. This nonconcentricity can occur and persist even without preexisting asymmetric cues, such as spindle placement or cellular adhesions. Cell-autonomous asymmetry is not explained by current models. We combined quantitative high-resolution live-cell microscopy with theoretical modeling to explore the mechanistic basis for asymmetric cytokinesis in the Caenorhabditis elegans zygote, with the goal of uncovering basic principles of ring closure. Our theoretical model suggests that feedback among membrane curvature, cytoskeletal alignment, and contractility is responsible for asymmetric cytokinetic furrowing. It also accurately predicts experimental perturbations of conserved ring proteins. The model further suggests that curvature-mediated filament alignment speeds up furrow closure while promoting energy efficiency. Collectively our work underscores the importance of membrane–cytoskeletal anchoring and suggests conserved molecular mechanisms for this activity.

Invadopodia and filopodia are dynamic, actin-based protrusions contributing to cancer cell migration, invasion, and metastasis. The force of actin bundles is essential for their protrusive activity. The bundling protein fascin is known to play a role in both invadopodia and filopodia. As it is more and more acknowledged that functionally related proteins cooperate, it is unlikely that only fascin bundles actin in these protrusions. Another interesting candidate is L-plastin, normally expressed in hematopoietic cells, but considered a common marker of many cancer types. We identified L-plastin as a new component of invadopodia, where it contributes to degradation and invasiveness. By means of specific, high-affinity nanobodies inhibiting bundling of fascin or L-plastin, we further unraveled their cooperative mode of action. We show that the bundlers cannot compensate for each other due to strikingly different bundling characteristics: L-plastin bundles are much thinner and less tightly packed. Composite bundles adopt an intermediate phenotype, with fascin delivering the rigidity and strength for protrusive force and structural stability, whereas L-plastin accounts for the flexibility needed for elongation. Consistent with this, elevated L-plastin expression promotes elongation and reduces protrusion density in cells with relatively lower L-plastin than fascin levels.

The path to the discovery of the actoclampins began with efforts to define profilin's role in actin-based pathogen and endosome rocketing. That research identified a set of FPPPP-containing cargo proteins and FPPPP-binding proteins that are consistently stationed within the polymerization zone during episodes of active motility. The very same biophysical clues that forced us to abandon Brownian Ratchet models guided us to the Actoclampin Hypothesis, which asserts that every propulsive filament possesses a (+)-end-tracking motor that generates the forces cells need to crawl. Each actoclampin motor is a multi-arm oligomeric complex, employing one arm to recruit/deliver Profilin•Actin•ATP to a growth-site located at the (+)-end of the lagging subfilament, while a second arm maintains an affinity-modulated binding interaction with the extreme (+)-end of the other subfilament. The alternating actions of these arms define a true molecular motor, the processivity of which explains why propelling filaments maintain full possession of their cargo. The Actoclampin Hypothesis also suggests how the energetics of tracker interactions with the (+)-end determines whether a given actoclampin is a passive (low force-producing) or active (high force-producing) motor, the latter requiring the Gibbs free energy of ATP hydrolysis. Another aim of this review is to acknowledge an earlier notional model that emerged from efforts to comprehend profilin's pivotal role(s) in actin-based cell motility.

Epithelia are essential barrier tissues that must be appropriately maintained for their correct function. To achieve this a plethora of protein interactions regulate epithelial cell number, structure and adhesion, and differentiation. Here we show that Cindr (the Drosophila Cin85 and Cd2ap ortholog) is required to maintain epithelial integrity. Reducing Cindr triggered cell delamination and movement. Most delaminating cells died. These behaviors were consistent with JNK activation previously associated with loss of epithelial integrity in response to ectopic oncogene activity. We confirmed a novel interaction between Cindr and Drosophila JNK (dJNK), which when perturbed caused inappropriate JNK signaling. Genetically reducing JNK signaling activity suppressed the effects of reducing Cindr. Furthermore, ectopic JNK signaling phenocopied loss of Cindr and was partially rescued by concomitant cindr over-expression. Thus, correct Cindr-dJNK stoichiometry is essential to maintain epithelial integrity and disturbing this balance may contribute to the pathogenesis of disease states, including cancer.

Polarized epithelia form by oriented cell divisions in which the mitotic spindle aligns parallel to the epithelial plane. To orient the mitotic spindle, cortical cues trigger the recruitment of NuMA-dynein-based motors, which pull on astral microtubules via the protein LGN. We demonstrate that the junctional protein Afadin is required for spindle orientation and correct epithelial morphogenesis of Caco-2 cysts. Molecularly, Afadin binds directly and concomitantly to F-actin and to LGN. We determined the crystallographic structure of human Afadin in complex with LGN and show that it resembles the LGN-NuMA complex. In mitosis, Afadin is necessary for cortical accumulation of LGN and NuMA above the spindle poles, in an F-actin-dependent manner. Collectively, our results depict Afadin as a molecular hub governing the enrichment of LGN and NuMA at the cortex. To our knowledge, Afadin is the first-described mechanical anchor between dynein and cortical F-actin.

Spectraplakins are crucially important communicators, linking cytoskeletal components to each other and cellular junctions. Microtubule actin crosslinking factor 1 (MACF1), also known as actin crosslinking family 7 (ACF7), is a member of the spectraplakin family. It is expressed in numerous tissues and cells as one extensively studied spectraplakin. MACF1 has several isoforms with unique structures and well-known function to be able to crosslink F-actin and microtubules. MACF1 is one versatile spectraplakin with various functions in cell processes, embryo development, tissue-specific functions, and human diseases. The importance of MACF1 has become more apparent in recent years. Here, we summarize the current knowledge on the presence and function of MACF1 and provide perspectives on future research of MACF1 based on our studies and others. [BMB Reports 2016; 49(1): 37-44]

Plastins are a family of actin binding proteins (ABPs) known to cross-link actin microfilaments in mammalian cells, creating actin microfilament bundles necessary to confer cell polarity and cell shape. Plastins also support cell movement in response to changes in environment, involved in cell/tissue growth and development. They also confer plasticity to cells and tissues in response to infection or other pathological conditions (e.g., inflammation). In the testis, the cell-cell anchoring junction unique to the testis that is found at the Sertoli cell-cell interface at the blood-testis barrier (BTB) and at the Sertoli-spermatid (e.g., 8-19 spermatids in the rat testis) is the basal and the apical ectoplasmic specialization (ES), respectively. The ES is an F-actin-rich anchoring junction constituted most notably by actin microfilament bundles. A recent report using RNAi that specifically knocks down plastin 3 has yielded some insightful information regarding the mechanism by which plastin 3 regulates the status of actin microfilament bundles at the ES via its intrinsic actin filament bundling activity. Herein, we provide a brief review on the role of plastins in the testis in light of this report, which together with recent findings in the field, we propose a likely model by which plastins regulate ES function during the epithelial cycle of spermatogenesis via their intrinsic activity on actin microfilament organization in the rat testis.

GRASP65 plays a role in Golgi ribbon formation. Because the gaps between Golgi stacks are heterogeneous and large, it is possible that other proteins may help GRASP65 in ribbon linking. Mena is a novel GRASP65-binding protein that promotes actin elongation and enhances GRASP65 oligomerization to link Golgi stacks into a ribbon., In mammalian cells, the Golgi reassembly stacking protein 65 (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers through the N-terminal GRASP domain. Because the GRASP domain is globular and relatively small, but the gaps between stacks are large and heterogeneous, it remains puzzling how GRASP65 physically links Golgi stacks into a ribbon. To explore the possibility that other proteins may help GRASP65 in ribbon linking, we used biochemical methods and identified the actin elongation factor Mena as a novel GRASP65-binding protein. Mena is recruited onto the Golgi membranes through interaction with GRASP65. Depleting Mena or disrupting actin polymerization resulted in Golgi fragmentation. In cells, Mena and actin were required for Golgi ribbon formation after nocodazole washout; in vitro, Mena and microfilaments enhanced GRASP65 oligomerization and Golgi membrane fusion. Thus Mena interacts with GRASP65 to promote local actin polymerization, which facilitates Golgi ribbon linking.

Item does not contain fulltext Podosomes are actin-rich adhesion structures that depend on Arp2/3-complex-based actin nucleation. We now report the identification of the formins FHOD1 and INF2 as novel components and additional actin-based regulators of podosomes in primary human macrophages. FHOD1 surrounds the podosome core and is also present at podosome-connecting cables, whereas INF2 localizes at the podosome cap structure. Using a variety of microscopy-based methods; including a semiautomated podosome reformation assay, measurement of podosome oscillations, FRAP analysis of single podosomes, and structured illumination microscopy, both formins were found to regulate different aspects of podosome-associated contractility, with FHOD1 mediating actomyosin contractility between podosomes, and INF2 regulating contractile events at individual podosomes. Moreover, INF2 was found to be a crucial regulator of podosome de novo formation and size. Collectively, we identify FHOD1 and INF2 as novel regulators of inter- and intra-structural contractility of podosomes. Podosomes thus present as one of the few currently identified structures which depend on the concerted activity of both Arp2/3 complex and specific formins and might serve as a model system for the analysis of complex actin architectures in cells.

Inturned-mediated complex formation of NPHP4 and DAAM1 is important for ciliogenesis and ciliary function in multiciliated cells, presumably because of its requirement for the local rearrangement of actin cytoskeleton., Motile cilia polarization requires intracellular anchorage to the cytoskeleton; however, the molecular machinery that supports this process remains elusive. We report that Inturned plays a central role in coordinating the interaction between cilia-associated proteins and actin-nucleation factors. We observed that knockdown of nphp4 in multiciliated cells of the Xenopus laevis epidermis compromised ciliogenesis and directional fluid flow. Depletion of nphp4 disrupted the subapical actin layer. Comparison to the structural defects caused by inturned depletion revealed striking similarities. Furthermore, coimmunoprecipitation assays demonstrated that the two proteins interact with each other and that Inturned mediates the formation of ternary protein complexes between NPHP4 and DAAM1. Knockdown of daam1, but not formin-2, resulted in similar disruption of the subapical actin web, whereas nphp4 depletion prevented the association of Inturned with the basal bodies. Thus, Inturned appears to function as an adaptor protein that couples cilia-associated molecules to actin-modifying proteins to rearrange the local actin cytoskeleton.

Microvilli are actin-based protrusions that amplify plasma membrane area and mediate interactions with the extracellular environment. We found that the multifunctional actin regulator cordon bleu promotes the growth of intestinal brush border microvilli. These results provide a new framework for investigating brush border biogenesis., Microvilli are actin-based protrusions found on the surface of diverse cell types, where they amplify membrane area and mediate interactions with the external environment. In the intestinal tract, these protrusions play central roles in nutrient absorption and host defense and are therefore essential for maintaining homeostasis. However, the mechanisms controlling microvillar assembly remain poorly understood. Here we report that the multifunctional actin regulator cordon bleu (COBL) promotes the growth of brush border (BB) microvilli. COBL localizes to the base of BB microvilli via a mechanism that requires its proline-rich N-terminus. Knockdown and overexpression studies show that COBL is needed for BB assembly and sufficient to induce microvillar growth using a mechanism that requires functional WH2 domains. We also find that COBL acts downstream of the F-BAR protein syndapin-2, which drives COBL targeting to the apical domain. These results provide insight into a mechanism that regulates microvillar growth during epithelial differentiation and have significant implications for understanding the maintenance of intestinal homeostasis.

The cooccurrence of Marfan syndrome and psychiatric disorders has been reported for many years. Furthermore, neuropsychological deficits have been shown to be associated with Marfan syndrome. The aim of the present article is to summarize findings from the sparse studies and case reports available. The results hold clinical and therapeutic implications and suggest that psychological and neuropsychological domains in Marfan syndrome patients should be carefully assessed. In particular, some patients may require specific rehabilitation programs. On this basis, a multidisciplinary approach to Marfan syndrome treatment seems mandatory.

Cellular actin assembly is controlled at the barbed ends of actin filaments, where capping protein (CP) limits polymerization. Twinfilin is a conserved in vivo binding partner of CP, yet the significance of this interaction has remained a mystery. Here, we discover that the C-terminal tail of Twinfilin harbors a CP-interacting (CPI) motif, identifying it as a novel CPI-motif protein. Twinfilin and the CPI-motif protein CARMIL have overlapping binding sites on CP. Further, Twinfilin binds competitively with CARMIL to CP, protecting CP from barbed-end displacement by CARMIL. Twinfilin also accelerates dissociation of the CP inhibitor V-1, restoring CP to an active capping state. Knockdowns of Twinfilin and CP each cause similar defects in cell morphology, and elevated Twinfilin expression rescues defects caused by CARMIL hyperactivity. Together, these observations define Twinfilin as the first ‘pro-capping’ ligand of CP and lead us to propose important revisions to our understanding of the CP regulatory cycle., eLife digest Plant and animal cells are supported by skeleton-like structures that can grow and shrink beneath the cell membrane, pushing and pulling on the edges of the cell. This scaffolding network – known as the cytoskeleton – contains long strands, or filaments, made from many identical copies of a protein called actin. The shape of the actin proteins allows them to slot together, end-to-end, and allows the strands to grow and shrink on-demand. When the strands are the correct length, the cell caps the growing ends with a protein known as Capping Protein. This helps to stabilize the cell’s skeleton, preventing the strands from getting any longer, or any shorter. Proteins that interfere with the activity of Capping Protein allow the actin strands to grow or shrink. Some, like a protein called V-1, attach to Capping Protein and get in the way so that it cannot sit on the ends of the actin strands. Others, like CARMIL, bind to Capping Protein and change its shape, making it more likely to fall off the strands. So far, no one had found a partner that helps Capping Protein limit the growth of the actin cytoskeleton. A protein called Twinfilin often appears alongside Capping Protein, but the two proteins seemed to have no influence on each other, and had what appeared to be different roles. Whilst Capping Protein blocks growth and stabilizes actin strands, Twinfilin speeds up their disassembly at their ends. But Johnston, Hilton et al. now reveal that the two proteins actually work together. Twinfilin helps Capping Protein resist the effects of CARMIL and V-1, and Capping Protein puts Twinfilin at the end of the strand. Thus, when Capping Protein is finally removed by CARMIL, Twinfilin carries on with disassembling the actin strands. The tail of the Twinfilin protein looks like part of the CARMIL protein, suggesting that they might interact with Capping Protein in the same way. Attaching a fluorescent tag to the Twinfilin tail revealed that the two proteins compete to attach to the same part of the Capping Protein. When mouse cells produced extra Twinfilin, it blocked the effects of CARMIL, helping to grow the actin strands. V-1 attaches to Capping Protein in a different place, but Twinfilin was also able to interfere with its activity. When Twinfilin attached to the CARMIL binding site, it did not directly block V-1 binding, but it made the protein more likely to fall off. Understanding how the actin cytoskeleton moves is a key question in cell biology, but it also has applications in medicine. Twinfilin plays a role in the spread of certain blood cancer cells, and in the formation of elaborate structures in the inner ear that help us hear. Understanding how Twinfilin and Capping Protein interact could open paths to new therapies for a range of medical conditions.

Cell protrusion is morphodynamically heterogeneous at the subcellular level. However, the mechanism of cell protrusion has been understood based on the ensemble average of actin regulator dynamics. Here, we establish a computational framework called HACKS (deconvolution of heterogeneous activity in coordination of cytoskeleton at the subcellular level) to deconvolve the subcellular heterogeneity of lamellipodial protrusion from live cell imaging. HACKS identifies distinct subcellular protrusion phenotypes based on machine-learning algorithms and reveals their underlying actin regulator dynamics at the leading edge. Using our method, we discover “accelerating protrusion”, which is driven by the temporally ordered coordination of Arp2/3 and VASP activities. We validate our finding by pharmacological perturbations and further identify the fine regulation of Arp2/3 and VASP recruitment associated with accelerating protrusion. Our study suggests HACKS can identify specific subcellular protrusion phenotypes susceptible to pharmacological perturbation and reveal how actin regulator dynamics are changed by the perturbation., Cell protrusion dynamics are heterogeneous at the subcellular level, but current analyses operate at the cellular or ensemble level. Here the authors develop a computational framework to quantify subcellular protrusion phenotypes and reveal the underlying actin regulator dynamics at the leading edge.

Background The familial hyperkalemic hypertension (FHHt) cullin 3 (CUL3) mutant does not degrade WNK kinases normally, thereby leading to thiazide-sensitive Na-Cl cotransporter (NCC) activation. CUL3 mutant (CUL3 Δ 9) does not bind normally to the COP9 signalosome (CSN), a deneddylase involved in regulating cullin-RING ligases. CUL3 Δ 9 also caused increased degradation of the CUL3-WNK substrate adaptor kelch-like 3 (KLHL3). Here, we sought to determine how defective CSN action contributes to the CUL3 Δ 9 phenotype. Methods The Pax8/LC1 mouse system was used to generate mice in which the catalytically active CSN subunit, Jab1 , was deleted only along the nephron, after full development (KS- Jab1 −/− ). Results Western blot analysis demonstrated that Jab1 deletion increased the abundance of neddylated CUL3. Moreover, total CUL3 expression was reduced, suggesting decreased CUL3 stability. KLHL3 was almost completely absent in KS- Jab1 −/− mice. Conversely, the protein abundances of WNK1, WNK4, and SPAK kinases were substantially higher. Activation of WNK4, SPAK, and OSR1 was indicated by higher phosphorylated protein levels and translocation of the proteins into puncta, as observed by immunofluorescence. The ratio of phosphorylated NCC to total NCC was also higher. Surprisingly, NCC protein abundance was low, likely contributing to hypokalemia and Na + and K + wasting. Additionally, long-term Jab1 deletion resulted in kidney damage. Conclusions Together, the results indicate that deficient CSN binding contributes importantly to the FHHt phenotype. Although defective CUL3 Δ 9-faciliated WNK4 degradation likely contributes, dominant effects on KLHL3 may be a second factor that is necessary for the phenotype.

Filopodia, dynamic membrane protrusions driven by polymerization of an actin filament core, can adhere to the extracellular matrix and experience both external and cell-generated pulling forces. The role of such forces in filopodia adhesion is however insufficiently understood. Here, we study filopodia induced by overexpression of myosin X, typical for cancer cells. The lifetime of such filopodia positively correlates with the presence of myosin IIA filaments at the filopodia bases. Application of pulling forces to the filopodia tips through attached fibronectin-coated laser-trapped beads results in sustained growth of the filopodia. Pharmacological inhibition or knockdown of myosin IIA abolishes the filopodia adhesion to the beads. Formin inhibitor SMIFH2, which causes detachment of actin filaments from formin molecules, produces similar effect. Thus, centripetal force generated by myosin IIA filaments at the base of filopodium and transmitted to the tip through actin core in a formin-dependent fashion is required for filopodia adhesion., Fiolopodia are involved in cell migration and their attachment to the ECM is mediated by integrin receptors. Here the authors show that myosin X induced filipodia adhesion to fibronectin requires activity of myosin IIA at the filopodium base and formin at the tip to support force transmission through the actin core.

Contractile muscle fibers produce enormous intrinsic forces during contraction/relaxation waves. These forces are directly applied to their cytoplasmic organelles including mitochondria, sarcoplasmic reticulum, and multiple nuclei. Data from our analysis of Drosophila larval somatic muscle fibers suggest that an intricate network of organized microtubules (MT) intermingled with Spectrin-Repeat-Containing Proteins (SRCPs) are major structural elements that protect muscle organelles and maintain their structure and position during muscle contraction. Whereas the perinuclear MT network provides structural rigidity to the myonucleus, the SRCPs Nesprin and Spectraplakin form semiflexible filamentous biopolymer networks, providing nuclei with the elasticity required to resist the contractile cytoplasmic forces produced by the muscle. Spectrin repeats are domains found in numerous structural proteins, which are able to unfold under tension and are subject to mechanical stresses in the cell. This unique composite scaffold combines rigidity and resilience in order to neutralize the oscillating cellular forces occurring during muscle contraction/relaxation waves and thereby protect myonuclei. We suggest that the elastic properties of SRCPs are critical for nuclear protection and proper function in muscle fibers.

Leiomodin (Lmod) is a class of potent tandem-G-actin-binding nucleators in muscle cells. Lmod mutations, deletion, or instability are linked to lethal nemaline myopathy. However, the lack of high-resolution structures of Lmod nucleators in action severely hampered our understanding of their essential cellular functions. Here we report the crystal structure of the actin-Lmod2162-495 nucleus. The structure contains two actin subunits connected by one Lmod2162-495 molecule in a non-filament-like conformation. Complementary functional studies suggest that the binding of Lmod2 stimulates ATP hydrolysis and accelerates actin nucleation and polymerization. The high level of conservation among Lmod proteins in sequence and functions suggests that the mechanistic insights of human Lmod2 uncovered here may aid in a molecular understanding of other Lmod proteins. Furthermore, our structural and mechanistic studies unraveled a previously unrecognized level of regulation in mammalian signal transduction mediated by certain tandem-G-actin-binding nucleators.

A little toxin can do a lot The actin cross-linking domain (ACD) is an actin-specific toxin produced by several bacterial pathogens. Heisler et al. discovered that ACD's pathogenic mechanism involves a highly unusual toxicity amplification cascade. Rather than directly inactivating the actin cytoskeleton, ACD blocks the activity of formins, actin regulatory proteins that play crucial roles in numerous cellular activities. ACD is exceptionally potent, even though its substrate is the most abundant protein of a eukaryotic cell: actin. Science , this issue p. 535

Excessive extracellular matrix deposition progressively replacing muscle fibres is the endpoint of most severe muscle diseases. Recent data indicate major involvement of microRNAs in regulating pro- and anti-fibrotic genes. To investigate the roles of miR-21 and miR-29 in muscle fibrosis in Duchenne muscle dystrophy, we evaluated their expression in muscle biopsies from 14 patients, and in muscle-derived fibroblasts and myoblasts. In Duchenne muscle biopsies, miR-21 expression was significantly increased, and correlated directly with COL1A1 and COL6A1 transcript levels. MiR-21 expression was also significantly increased in Duchenne fibroblasts, more so after TGF-β1 treatment. In Duchenne fibroblasts the expression of miR-21 target transcripts PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SPRY-1 (Sprouty homolog 1) was significantly reduced; while collagen I and VI transcript levels and soluble collagen production were significantly increased. MiR-29a and miR-29c were significantly reduced in Duchenne muscle and myoblasts, and miR-29 target transcripts, COL3A1, FBN1 and YY1, significantly increased. MiR-21 silencing in mdx mice reduced fibrosis in the diaphragm muscle and in both Duchenne fibroblasts and mdx mice restored PTEN and SPRY-1 expression, and significantly reduced collagen I and VI expression; while miR-29 mimicking in Duchenne myoblasts significantly decreased miR-29 target transcripts. These findings indicate that miR-21 and miR-29 play opposing roles in Duchenne muscle fibrosis and suggest that pharmacological modulation of their expression has therapeutic potential for reducing fibrosis in this condition. copy; 2015 .

Reactive astrocytes and activated microglia are the key players in several pathophysiologic modifications of the central nervous system. We used the spared nerve injury (SNI) of the sciatic nerve to induce glial maladaptive response in the ventral horn of lumbar spinal cord and examine its role in the remodeling of the tripartite synapse plasticity. Imaging the ventral horn revealed that SNI was associated with both an early microglial and astrocytic activation, assessed, respectively, by analysis of Iba1 and GFAP expression. Microglia, in particular, localized peculiarly surrounding the motor neurons somata. Perineuronal astrocytes, which play a key role in maintaining the homeostasis of neuronal circuitry, underwent a substantial phenotypic change following peripheral axotomy, producing reactive gliosis. The gliosis was associated with the reduction of glial aminoacid transporters (GLT1 and GlyT1) and increase of neuronal glutamate transporter EAAC1. Although the expression of GABAergic neuronal marker GAD65/67 showed no change, glutamate increase, as demonstrated by HPLC analysis, shifted the excitatory/inhibitory balance as showed by the net increase of the glutamate/GABA ratio. Moreover, endogenous NGF levels were altered in SNI animals and not restored by the intrathecal NGF administration. This treatment reverted phenotypic changes associated with reactive astrocytosis, but failed to modify microglia activation. These findings on one hand confirm the correlation between gliopathy and maladaptive plasticity of the spinal synaptic circuitry, on the other hand add new data concerning the complex peculiar behavior of different glial cells in neuronal degenerative processes, defining a special role of microglia in sustaining the inflammatory response.

International audience; Turnover of actin networks in cells requires the fast disassembly of aging actin structures. While ADF/cofilin and Aip1 have been identified as central players, how their activities are modulated by the architecture of the networks remains unknown. Using our ability to reconstitute a diverse array of cellular actin organizations, we found that ADF/cofilin binding and ADF/cofilin-mediated disassembly both depend on actin geometrical organization. ADF/cofilin decorates strongly and stabilizes actin cables, whereas its weaker interaction to Arp2/3 complex networks is correlated with their dismantling and their reorganization into stable architectures. Cooperation of ADF/cofilin with Aip1 is necessary to trigger the full disassembly of all actin filament networks. Additional experiments performed at the single-molecule level indicate that this cooperation is optimal above a threshold of 23 molecules of ADF/cofilin bound as clusters along an actin filament. Our results indicate that although ADF/cofilin is able to dismantle selectively branched networks through severing and debranching, stochastic disassembly of actin filaments by ADF/cofilin and Aip1 represents an efficient alternative pathway for the full disassembly of all actin networks. Our data support a model in which the binding of ADF/cofilin is required to trigger a structural change of the actin filaments, as a prerequisite for their disassembly by Aip1.

The present review examines the role of actin binding proteins (ABPs) on blood-testis barrier (BTB), an androgen-dependent ultrastructure in the testis, in particular their involvement on BTB remodeling during spermatogenesis.The BTB divides the seminiferous epithelium into the basal and the adluminal compartments. The BTB is constituted by coexisting actin-based tight junction, basal ectoplasmic specialization, and gap junction, and also intermediate filament-based desmosome between Sertoli cells near the basement membrane. Junctions at the BTB undergo continuous remodeling to facilitate the transport of preleptotene spermatocytes residing in the basal compartment across the immunological barrier during spermatogenesis. Thus, meiosis I/II and postmeiotic spermatid development take place in the adluminal compartment behind the BTB. BTB remodeling also regulates exchanges of biomolecules between the two compartments. As tight junction, basal ectoplasmic specialization, and gap junction use F-actin for attachment, actin microfilaments rapidly convert between their bundled and unbundled/branched configuration to confer BTB plasticity. The events of actin reorganization are regulated by two major classes of ABPs that convert actin microfilaments between their bundled and branched/unbundled configuration.We provide a model on how ABPs regulate BTB remodeling, shedding new light on unexplained male infertility, such as environmental toxicant-induced reproductive dysfunction since the testis, in particular the BTB, is sensitive to environmental toxicants, such as cadmium, bisphenol A, phthalates, and PFOS (perfluorooctanesulfonic acid or perfluorooctane sulfonate).

Summary Cell-shape changes associated with processes like cytokinesis and motility proceed on several-second timescales but are derived from molecular events, including protein-protein interactions, filament assembly, and force generation by molecular motors, all of which occur much faster [1–4]. Therefore, defining the dynamics of such molecular machinery is critical for understanding cell-shape regulation. In addition to signaling pathways, mechanical stresses also direct cytoskeletal protein accumulation [5–7]. A myosin-II-based mechanosensory system controls cellular contractility and shape during cytokinesis and under applied stress [6, 8]. In Dictyostelium , this system tunes myosin II accumulation by feedback through the actin network, particularly through the crosslinker cortexillin I. Cortexillin-binding IQGAPs are major regulators of this system. Here, we defined the short timescale dynamics of key cytoskeletal proteins during cytokinesis and under mechanical stress, using fluorescence recovery after photobleaching and fluorescence correlation spectroscopy, to examine the dynamic interplay between these proteins. Equatorially enriched proteins including cortexillin I, IQGAP2, and myosin II recovered much more slowly than actin and polar crosslinkers. The mobility of equatorial proteins was greatly reduced at the furrow compared to the interphase cortex, suggesting their stabilization during cytokinesis. This mobility shift did not arise from a single biochemical event, but rather from a global inhibition of protein dynamics by mechanical-stress-associated changes in the cytoskeletal structure. Mechanical tuning of contractile protein dynamics provides robustness to the cytoskeletal framework responsible for regulating cell shape and contributes to cytokinesis fidelity.

Accurate regulation of microfilament dynamics is central to cell growth, motility and response to environmental stimuli. Stabilizing and depolymerizing proteins control the steady-state levels of filamentous (F-) actin. Capping protein (CP) binds to free barbed ends, thereby arresting microfilament growth and restraining elongation to remaining free barbed ends. In all CPs characterized to date, alpha and beta subunits form the active heterodimer. Here, we show in a eukaryotic parasitic cell that the two CP subunits can be functionally separated. Unlike the beta subunit, the CP alpha subunit of the apicomplexan parasite Plasmodium is refractory to targeted gene deletion during blood infection in the mammalian host. Combinatorial complementation of Plasmodium berghei CP genes with the orthologs from Plasmodium falciparum verified distinct activities of CP alpha and CP alpha/beta during parasite life cycle progression. Recombinant Plasmodium CP alpha could be produced in Escherichia coli in the absence of the beta subunit and the protein displayed F-actin capping activity. Thus, the functional separation of two CP subunits in a parasitic eukaryotic cell and the F-actin capping activity of CP alpha expand the repertoire of microfilament regulatory mechanisms assigned to CPs.

Dynamic reorganization of the cortical cytoskeleton is essential for numerous cellular processes, including B- and T-cell activation and migration. The ezrin-radixin-moesin (ERM) family of proteins plays structural and regulatory roles in the rearrangement of plasma membrane flexibility and protrusions through its members' reversible interaction with cortical actin filaments and the plasma membrane. Recent studies demonstrated that ERM proteins not only are involved in cytoskeletal organization but also offer a platform for the transmission of signals in response to a variety of extracellular stimuli through their ability to cross-link transmembrane receptors with downstream signaling components. In this review, we summarize present knowledge relating to ERMs and recent progress made toward elucidating a novel role for them in the regulation of B-cell function in health and disease.

Reorganization of the actin cytoskeleton is essential for cell motility and chemotaxis. Actin-binding proteins (ABPs) and membrane lipids, especially phosphoinositides PI(4,5)P2 and PI(3,4,5)P3 are involved in the regulation of this reorganization. At least 15 ABPs have been reported to interact with, or regulated by phosphoinositides (PIPs) whose synthesis is regulated by extracellular signals. Recent studies have uncovered several parallel intracellular signalling pathways that crosstalk in chemotaxing cells. Here, we review the roles of ABPs and phosphoinositides in chemotaxis and cell migration. Linked Articles This article is part of a themed section on Cytoskeleton, Extracellular Matrix, Cell Migration, Wound Healing and Related Topics. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-24

Neuronal migration and subsequent differentiation play critical roles for establishing functional neural circuitry in the developing brain. However, the molecular mechanisms that regulate these processes are poorly understood. Here, we show that microtubule actin crosslinking factor 1 (MACF1) determines neuronal positioning by regulating microtubule dynamics and mediating GSK-3 signaling during brain development. First, using MACF1 floxed allele mice and in utero gene manipulation, we find that MACF1 deletion suppresses migration of cortical pyramidal neurons and results in aberrant neuronal positioning in the developing brain. The cell autonomous deficit in migration is associated with abnormal dynamics of leading processes and centrosomes. Furthermore, microtubule stability is severely damaged in neurons lacking MACF1, resulting in abnormal microtubule dynamics. Finally, MACF1 interacts with and mediates GSK-3 signaling in developing neurons. Our findings establish a cellular mechanism underlying neuronal migration and provide insights into the regulation of cytoskeleton dynamics in developing neurons.

The intracellular environment is composed of a filamentous network that exhibits dynamic turnover of cytoskeletal components and internal force generation from molecular motors. Particle tracking microrheology enables a means to probe the internal mechanics and dynamics. Here, we develop an analytical model to capture the basic features of the active intracellular mechanical environment, including both thermal and motor-driven effects, and show consistency with a diverse range of experimental microrheology data. We further perform microrheology experiments, integrated with Brownian dynamics simulations of the active cytoskeleton, on metastatic breast cancer cells embedded in a three-dimensional collagen matrix with and without the presence of epidermal growth factor to probe the intracellular mechanical response in a physiologically mimicking scenario. Our results demonstrate that EGF stimulation can alter intracellular stiffness and power output from molecular motor-driven fluctuations in cells overexpressing an invasive isoform of the actin-associated protein Mena.

Intranuclear rods are aggregates consisting of actin and cofilin that are formed in the nucleus in consequence of chemical or mechanical stress conditions. The formation of rods is implicated in a variety of pathological conditions, such as certain myopathies and some neurological disorders. It is still not well understood what exactly triggers the formation of intranuclear rods, whether other proteins are involved, and what the underlying mechanisms of rod assembly or disassembly are. In this study, Dictyostelium discoideum was used to examine appearance, stages of assembly, composition, stability, and dismantling of rods. Our data show that intranuclear rods, in addition to actin and cofilin, are composed of a distinct set of other proteins comprising actin-interacting protein 1 (Aip1), coronin (CorA), filactin (Fia), and the 34 kDa actin-bundling protein B (AbpB). A finely tuned spatio-temporal pattern of protein recruitment was found during formation of rods. Aip1 is important for the final state of rod compaction indicating that Aip1 plays a major role in shaping the intranuclear rods. In the absence of both Aip1 and CorA, rods are not formed in the nucleus, suggesting that a sufficient supply of monomeric actin is a prerequisite for rod formation.

Marine snail egg capsules are shock-absorbing bioelastomers made from precursor “egg case proteins” (ECPs) that initially lack long-range order. During capsule formation, these proteins self-assemble into coiled-coil filaments that subsequently align into microscopic layers, a multiscale process which is crucial to the capsules’ shock-absorbing properties. In this study, we show that the self-assembly of ECPs into their functional capsule material is mediated by a bundling protein that facilitates the aggregation of coiled-coil building blocks and their gelation into a prefinal capsule prior to final stabilization. This low molecular weight bundling protein, termed Pugilina cochlidium Bundling Protein (PcBP), led to gelation of native extracts from gravid snails, whereas crude extracts lacking PcBP did not gelate and remained as a protein solution. Refolding and reconcentration of recombinant PcBP induced bundling and aggregation of ECPs, as evidenced by ECPs oligomerization. We propose that the secretion of PcBP in vivo is a time-specific event during the embryo encapsulation process prior to cross-linking in the ventral pedal gland (VPG). Using molecular dynamics (MD) simulations, we further propose plausible disulfide binding sites stabilizing two PcBP monomers, as well as a polarized surface charge distribution, which we suggest plays an important role in the bundling mechanism. Overall, this study shows that controlled bundling is a key step during the extra-cellular self-assembly of egg capsules, which has previously been overlooked. NRF (Natl Research Foundation, S’pore) ASTAR (Agency for Sci., Tech. and Research, S’pore) MOE (Min. of Education, S’pore)