Actin Cross-linking Research Articles

Binding Dynamics of α-Actinin-4 in Dependence of Actin Cortex Tension

Mechanosensation of cells is an important prerequisite for cellular function, e.g., in the context of cell migration, tissue organization, and morphogenesis. An important mechanochemical transducer is the actin cytoskeleton. In fact, previous studies have shown that actin cross-linkers such as α-actinin-4 exhibit mechanosensitive properties in their binding dynamics to actin polymers. However, to date, a quantitative analysis of tension-dependent binding dynamics in live cells is lacking. Here, we present a, to our knowledge, new technique that allows us to quantitatively characterize the dependence of cross-linking lifetime of actin cross-linkers on mechanical tension in the actin cortex of live cells. We use an approach that combines parallel plate confinement of round cells, fluorescence recovery after photobleaching, and a mathematical mean-field model of cross-linker binding. We apply our approach to the actin cross-linker α-actinin-4 and show that the cross-linking time of α-actinin-4 homodimers increases approximately twofold within the cellular range of cortical mechanical tension, rendering α-actinin-4 a catch bond in physiological tension ranges.

Comprehensive analysis of protein expression levels and phosphorylation levels in host skin in response to tick (Haemaphysalis longicornis) bite

Ticks are parasitic arthropods that suck blood from the surface of most vertebrates. They can transmit a variety of pathogens. The blood sucking of ticks causes varying degrees of damage to the skin of the host. Proteins related to immune regulation, vascular repair, and wound healing in mammalian skin respond to tick bites by regulating their expression and post-translational modifications to protect the skin from injury. Phosphorylation of proteins, as the most common post-translational modification of proteins, plays an important role in the rapid regulation of cell signal transduction, gene expression and cell cycle. To systematically explore the molecular regulatory mechanisms employed by mammalian skin to resist tick bites, larval, nymphal, and adult Haemaphysalis longicornis were used to bite the skin tissues of healthy rabbits in the present study. The quantitative proteomic technology data-independent acquisition was then carried out to investigate in depth the changes in protein expression and phosphorylation in rabbit skin after tick bite. The results showed that among the 4034 proteins and 1795 phosphorylated proteins identified, a total of 202 proteins and 435 phosphorylation sites were changed after H. longicornis bite. In order to provide convenience for sucking blood, active substances in the saliva of H. longicornis injected into the rabbit's skin can cause the expression level of trichohyalin and peptidyl arginine deiminase 3 in the skin of the host downregulate, which can make the host hair loss and regeneration disorders. At the same time, the active substances in saliva of the H. longicornis led to the phosphorylation of microtubule actin cross-linking factor 1 in the host's skin and further inactivation, so as to delay the healing of the host wound. In response to tick bites, the host skin promotes coagulation through high expression of fibrinogen and fibronectin, and vascular repair through high expression of integrin linked kinase and tenascin C, as well as accelerated phosphorylation of the phosphorylated protein Nck adaptor protein 1, and wound healing through high expression of ezrin and integrin. The upregulation of proteins such as coronin, NADPH oxidase, calnexin, and calreticulin and phosphorylation level of IL-4R in the host skin after the H. longicornis bite indicated that the immune response was playing an important defensive role in response to tick bites. Meanwhile, we found that the upregulated two lectins, mannose receptor C-type 1 and DC-SIGN, may serve as molecular makers to identify and monitor whether the skin is bitten by ticks. SignificanceHaemaphysalis longicornis are parasitic arthropods that suck blood from the surface of most vertebrates. They can transmit a variety of pathogens and are harmful to humans and livestock.The present study is the first quantitative proteomic study on protein expression levels in the rabbit skin after infection by H. longicornis. It is also the first quantitative phosphoproteomic study in the host skin infected by ticks. In this study, we found that tick bites cause the host hair loss and regeneration disorders. For resisting tick bite, the host activates the immune response and initiates vascular repair and wound-healing systems. In addition, some phosphorylated proteins promote host immunity and vascular repair. These results can help us further understand the defence mechanism of the host against tick bites, provide a basis for the development of an anti-tick vaccine, the development of anti-tick drugs, and the diagnosis of tick-borne diseases.

Dynamic actin cross-linking governs the cytoplasm's transition to fluid-like behavior.

Cells precisely control their mechanical properties to organize and differentiate into tissues. The architecture and connectivity of cytoskeletal filaments change in response to mechanical and biochemical cues, allowing the cell to rapidly tune its mechanics from highly cross-linked, elastic networks to weakly cross-linked viscous networks. While the role of actin cross-linking in controlling actin network mechanics is well-characterized in purified actin networks, its mechanical role in the cytoplasm of living cells remains unknown. Here, we probe the frequency-dependent intracellular viscoelastic properties of living cells using multifrequency excitation and in situ optical trap calibration. At long timescales in the intracellular environment, we observe that the cytoskeleton becomes fluid-like. The mechanics are well-captured by a model in which actin filaments are dynamically connected by a single dominant cross-linker. A disease-causing point mutation (K255E) of the actin cross-linker α-actinin 4 (ACTN4) causes its binding kinetics to be insensitive to tension. Under normal conditions, the viscoelastic properties of wild-type (WT) and K255E+/– cells are similar. However, when tension is reduced through myosin II inhibition, WT cells relax 3× faster to the fluid-like regime while K255E+/– cells are not affected. These results indicate that dynamic actin cross-linking enables the cytoplasm to flow at long timescales.

Phosphorylation of ACTN4 Leads to Podocyte Vulnerability and Proteinuric Glomerulosclerosis.

Genetic mutations in α-actinin-4 (ACTN4)-an important actin crosslinking cytoskeletal protein that provides structural support for kidney podocytes-have been linked to proteinuric glomerulosclerosis in humans. However, the effect of post-translational modifications of ACTN4 on podocyte integrity and kidney function is not known. Using mass spectrometry, we found that ACTN4 is phosphorylated at serine (S) 159 in human podocytes. We used phosphomimetic and nonphosphorylatable ACTN4 to comprehensively study the effects of this phosphorylation in vitro and in vivo. We conducted x-ray crystallography, F-actin binding and bundling assays, and immunofluorescence staining to evaluate F-actin alignment. Microfluidic organ-on-a-chip technology was used to assess for detachment of podocytes simultaneously exposed to fluid flow and cyclic strain. We then used CRISPR/Cas9 to generate mouse models and assessed for renal injury by measuring albuminuria and examining kidney histology. We also performed targeted mass spectrometry to determine whether high extracellular glucose or TGF-β levels increase phosphorylation of ACTN4. Compared with the wild type ACTN4, phosphomimetic ACTN4 demonstrated increased binding and bundling activity with F-actin in vitro. Phosphomimetic Actn4 mouse podocytes exhibited more spatially correlated F-actin alignment and a higher rate of detachment under mechanical stress. Phosphomimetic Actn4 mice developed proteinuria and glomerulosclerosis after subtotal nephrectomy. Moreover, we found that exposure to high extracellular glucose or TGF-β stimulates phosphorylation of ACTN4 at S159 in podocytes. These findings suggest that increased phosphorylation of ACTN4 at S159 leads to biochemical, cellular, and renal pathology that is similar to pathology resulting from human disease-causing mutations in ACTN4. ACTN4 may mediate podocyte injury as a consequence of both genetic mutations and signaling events that modulate phosphorylation.

Roles of Fascin mRNA expression in colorectal cancer: Meta-analysis and bioinformatics analysis.

Fascin (encoded by FSCN1) is a globular actin cross-linking protein that is required for the formation of actin-based cell surface processes, which are critical for cell migration and cell-matrix adhesion. In the present study, a systematic meta-analysis and bioinformatics analysis was used to identify clinicopathological or prognostic parameters in patients with colorectal cancer. A total of 17 articles were included in the present study obtained from PubMed, Web of Science, Wanfang data, SinoMed and CNKI databases. Odd ratios (ORs) and the corresponding 95% confidence intervals (CIs) were used to estimate the prognostic significance of Fascin expression in patients with colorectal cancer, and the association between Fascin expression and clinicopathological factors. There was a significant correlation between high Fascin expression and poor overall survival rates in patients with colorectal cancer (OR=0.48; 95% CI, 0.38-0.60; P<0.000001). The meta-analysis showed that the expression of Fascin was significantly higher in colorectal cancer tissue compared with the normal mucosa (OR=0.13; 95% CI, 0.10-0.16; P<0.000001) and adenoma (OR=0.23; 95% CI, 0.15-0.34; P<0.000001). Fascin expression was also associated with depth of invasion (OR=0.31; 95% CI, 0.19-0.50; P<0.000001), lymph node metastasis (OR=3.07; 95% CI, 1.72-5.46; P=0.0001), Dukes stage (OR=0.14; 95% CI, 0.04-0.46; P=0.001), Tumor-Node-Metastasis stage (OR=0.38; 95% CI, 0.21-0.71; P=0.003) and dedifferentiation (OR=0.42; 95% CI, 0.19-0.94; P=0.04). According to the bioinformatics analyses, FSCN1 mRNA expression levels were higher in colorectal cancer and adenoma tissues compared with the normal tissues (P<0.05). According to TCGA, FSCN1 mRNA expression was associated with a less favorable prognosis in patients with colorectal cancer as an independent factor (P<0.05), and positively correlated with depth of invasion, microsatellite instability and low serum carcinoembryonic antigen levels in colorectal cancer. Taken together, the results of the present study suggested that Fascin expression is a potential marker of tumorigenesis, aggressiveness and poor prognosis in patients with colorectal cancer.

Abstract B070: Loss of cytoskeletal protein palladin desensitizes pancreatic cancer associated fibroblast to TGFβ1-dependent desmoplastic induction

Abstract Pancreatic ductal adenocarcinoma (PDAC) claims ~93% of patient lives in less than 5 years proceeding diagnosis. Therapeutic options for PDAC are challenged by the deposition of a fibrous-like extracellular matrix (ECM) known as desmoplasia. This desmoplastic-ECM (D-ECM) forms the bulk of the tumor mass while remodeling the once tumor-suppressive microenvironment to become tumor permissive. Ironically, ablation of the D-ECM results in an even more aggressive PDAC. Thus, unraveling the mechanism of D-ECM induction is vital to reinstating the natural tumor-restrictive microenvironment of the pancreas. Although the underlying biology remains unclear, the D-ECM is initiated and maintained by activated cancer associated fibroblasts (CAFs). CAF activation is primarily driven by TGFβ1 signaling, which causes cytoskeletal rearrangements in support of CAF-dependent D-ECM onset. As such, the goal of this study is to define the cytoskeletal regulation needed to restore the natural tumor-suppressive properties of the pancreas. Therefore, we focused on the actin cross-linker palladin whose expression is regulated by TGFβ1 and has been identified as an independent prognostic marker for PDAC progression as well as CAF activation. We postulate palladin has an isoform-specific role in PDAC-associated desmoplasia that ultimately fuels cancer progression. Using an in vivo-mimetic 3D stroma model, I used a CRISPR/Cas9 approach to delete one or both of the major palladin isoforms in patient-derived CAFs and assessed CAF's activation via quantitative immunoblots, real-time polymerase chain reaction, and multiplex confocal microscopy in addition to palladin knockout CAF-derived ECM's ability to influence PDAC cell viability and invasive spread. My data indicate that palladin isoforms 3 and 4 are required for TGFβ1-depdendent CAF activation as well as for CAF-dependent D-ECM induction whereby knockdown of either isoform in CAFs results in a normalized ECM that restrains the inherent aggressive nature of PDAC cells. These findings suggest that targeting palladin alters the desmoplastic compartment by impeding tumor progression. Taken together, these results propose a strategy to restore a normal tumor-suppressive stroma in PDAC that will complement current PDAC treatment dogmas. Citation Format: Jennifer Alexander, Edna Cukierman. Loss of cytoskeletal protein palladin desensitizes pancreatic cancer associated fibroblast to TGFβ1-dependent desmoplastic induction [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B070.

Tea Polyphenols Affect Oxidative Modification and Solution Stability of Myofibrillar Protein from Grass Carp (Ctenopharyngodon idellus)

This study investigated the effects of different concentrations (0, 5, 10, 20, 50, and 100 μmol/g protein) of tea polyphenols (TP) on the oxidative modification and the physicochemical, structural, and gelling properties of myofibrillar protein (MP) from grass carp (Ctenopharyngodon idellus) oxidized by a hydroxyl radical-generating system. The results showed that low concentrations (5 and 10 μmol/g protein) of TP could effectively inhibit the formation of carbonyl groups and dityrosine, the loss of sulfhydryl groups and α-helix conformation, and the change of the tertiary structure of MP caused by hydroxyl radicals. Moreover, the presence of TP in all concentrations decreased the surface hydrophobicity of MP. TP at 10 μmol/g protein had better effects on preventing the oxidation-induced cross-linking and aggregation of myosin heavy chain (MHC) and actin of MP, keeping the stability of MP solutions with lower turbiscan stability index (TSI) values, and improving gelling properties characterized by higher hardness and gel strength of MP gels. By contrast, excessive presence of TP (20, 50, and 100 μmol/g protein) showed pro-oxidative effects on oxidatively stressed MP, which was detrimental to the MP and contributed to the denaturation and irregular aggregation of MP, the loss of MP solution stability, and lower gelling capacity with poor texture and gel strength. The concentration-dependent effects of TP on MP depended on the extent of MP oxidative modification and MP-TP interactions, indicating that a proper amount of TP has the potential to protect MP from oxidation and to enhance the gelling capacity of surimi during processing.

On the mechanical response of the actomyosin cortex during cell indentations.

A mechanical model is presented to analyze the mechanics and dynamics of the cell cortex during indentation. We investigate the impact of active contraction on the cross-linked actin network for different probe sizes and indentation rates. The essential molecular mechanisms of filament stretching, cross-linking and motor activity, are represented by an active and viscous mechanical continuum. The filaments behave as worm-like chains linked either by passive rigid linkers or by myosin motors. In the first example, the effects of probe size and loading rate are evaluated using the model for an idealized rounded cell shape in which properties are based on the results of parallel-plate rheometry available in the literature. Extreme cases of probe size and indentation rate are taken into account. Afterward, AFM experiments were done by engaging smooth muscle cells with both sharp and spherical probes. By inverse analysis with finite element software, our simulations mimicking the experimental conditions show the model is capable of fitting the AFM data. The results provide spatiotemporal dependence on the size and rate of the mechanical stimuli. The model captures the general features of the cell response. It characterizes the actomyosin cortex as an active solid at short timescales and as a fluid at longer timescales by showing (1) higher levels of contraction in the zones of high curvature; (2) larger indentation forces as the probe size increases; and (3) increase in the apparent modulus with the indentation depth but no dependence on the rate of the mechanical stimuli. The methodology presented in this work can be used to address and predict microstructural dependence on the force generation of living cells, which can contribute to understanding the broad spectrum of results in cell experiments.

Bond Type and Discretization of Nonmuscle Myosin II Are Critical for Simulated Contractile Dynamics

Molecular motors drive cytoskeletal rearrangements to change cell shape. Myosins are the motors that move, cross-link, and modify the actin cytoskeleton. The primary force generator in contractile actomyosin networks is nonmuscle myosin II (NMMII), a molecular motor that assembles into ensembles that bind, slide, and cross-link actin filaments (F-actin). The multivalence of NMMII ensembles and their multiple roles have confounded the resolution of crucial questions, including how the number of NMMII subunits affects dynamics and what affects the relative contribution of ensembles’ cross-linking versus motoring activities. Because biophysical measurements of ensembles are sparse, modeling of actomyosin networks has aided in discovering the complex behaviors of NMMII ensembles. Myosin ensembles have been modeled via several strategies with variable discretization or coarse graining and unbinding dynamics, and although general assumptions that simplify motor ensembles result in global contractile behaviors, it remains unclear which strategies most accurately depict cellular activity. Here, we used an agent-based platform, Cytosim, to implement several models of NMMII ensembles. Comparing the effects of bond type, we found that ensembles of catch-slip and catch motors were the best force generators and binders of filaments. Slip motor ensembles were capable of generating force but unbound frequently, resulting in slower contractile rates of contractile networks. Coarse graining of these ensemble types from two sets of 16 motors on opposite ends of a stiff rod to two binders, each representing 16 motors, reduced force generation, contractility, and the total connectivity of filament networks for all ensemble types. A parallel cluster model, previously used to describe ensemble dynamics via statistical mechanics, allowed better contractility with coarse graining, though connectivity was still markedly reduced for this ensemble type with coarse graining. Together, our results reveal substantial tradeoffs associated with the process of coarse graining NMMII ensembles and highlight the robustness of discretized catch-slip ensembles in modeling actomyosin networks.

Ezrin protects bovine spermatozoa from spontaneous acrosome reaction

To interact and penetrate the egg, the spermatozoon must undergo a maturation step called the acrosome reaction (AR) in close proximity to the egg. This process can take place only after a series of biochemical changes to the sperm occur in the female reproductive tract, collectively called capacitation. Spermatozoa can undergo spontaneous-acrosome reaction (sAR) before reaching the vicinity of the egg, preventing successful fertilization. Several mechanisms were shown to protect spermatozoa from undergoing sAR. Here we describe the involvement of the actin cross-linker, Ezrin in the mechanism that protects spermatozoa from sAR. Inhibition of Ezrin stimulates sAR and inhibits actin polymerization. Ezrin is highly phosphorylated/activated during the first hour of the capacitation process, and its phosphorylation rate is subsequently decreased. Ezrin phosphorylation depends on protein kinase A (PKA) and calmodulin kinase II (CaMKII) activities, and to some extent on phosphatidyl-inositol-4-kinase (PI4K) activity. Inhibition of these three kinases stimulates sAR, in which the effect of PI4K inhibition, but not PKA or CaMKII inhibition, can be reversed by increasing p-Ezrin using a phosphatase inhibitor. All together, we showed that three kinases mediate Ezrin activation during spermatozoa capacitation, leading to actin polymerization in a mechanism that prevents sAR.

In‐frame Genetic Disruption of SPECC1L Microtubule‐Interaction Domain Causes Embryonic Tissue Movement and Fusion Defects

Movement and fusion events are critical during embryonic development; defects in these events lead to common birth anomalies. For example, failure of neural tube, secondary palate and ventral body wall closure leads to exencephaly, cleft palate, and omphalocele, respectively. Autosomal‐dominant SPECC1L mutations have been identified in patients with syndromic malformations, including hypertelorism, cleft palate and omphalocele. These mutations cluster in the second coiled‐coil (CCD2) and calponin homology (CHD) domains, which interact with microtubule and actin, respectively. To study SPECC1L function in mice, we first generated genomic deletions that resulted in out‐of‐frame truncations. Homozygous mutants for these truncations, including those lacking CCD2 and CHD, died shortly after birth without cleft palate or omphalocele. We then generated a series of targeted in‐frame deletions in CCD2, homozygotes for which also exhibited perinatal lethality but now with ~50% exencephaly, ~50% cleft palate, and ~50% omphalocele. While the omphalocele phenotype segregated equally with the other two phenotypes, exencephaly and cleft palate were never observed in the same embryo, suggesting antagonistic tissue mechanics between neural tube and palate closure. Consistently, we found that the oral cavity was narrower in mutant embryos with exencephaly, allowing for the palatal shelves to close eventually. We have shown previously that SPECC1L deficiency leads to impaired cell speed and directionality. We now show that subcellular SPECC1L‐ΔCCD2 protein distribution was abnormal, accumulating asymmetrically and associating almost exclusively with filamentous actin. Thus, loss of SPECC1L interaction with microtubules – while maintaining actin association – was more detrimental to cell and tissue movement than loss of SPECC1L protein entirely. Taken together, our results showed that in‐frame perturbations of CCD2 in SPECC1L protein specifically affect embryonic closure of several tissues, suggesting that human SPECC1L CCD2 missense mutations are gain‐of‐function. Our data also revealed a novel role for SPECC1L cross‐linking of actin and microtubule cytoskeletons in embryonic tissue movement and fusion events.Support or Funding InformationThis project was supported in part by the National Institutes of Health grants DE026172 (I.S.). Core support and pilot funding was also provided by National Institutes of Health Center of Biomedical Research Excellence (COBRE) grant (P20 GM104936), Kansas IDeA Network for Biomedical Research Excellence (INBRE) grant (P20 GM103418), and Kansas Intellectual and Developmental Disabilities Research Center (IDDRC) grant (P30 HD 002528).

Magnaporthe oryzae fimbrin organizes actin networks in the hyphal tip during polar growth and pathogenesis.

Magnaporthe oryzae causes rice blast disease, but little is known about the dynamic restructuring of the actin cytoskeleton during its polarized tip growth and pathogenesis. Here, we used super-resolution live-cell imaging to investigate the dynamic organization of the actin cytoskeleton in M. oryzae during hyphal tip growth and pathogenesis. We observed a dense actin network at the apical region of the hyphae and actin filaments originating from the Spitzenkörper (Spk, the organizing center for hyphal growth and development) that formed branched actin bundles radiating to the cell membrane. The actin cross-linking protein Fimbrin (MoFim1) helps organize this actin distribution. MoFim1 localizes to the actin at the subapical collar, the actin bundles, and actin at the Spk. Knockout of MoFim1 resulted in impaired Spk maintenance and reduced actin bundle formation, preventing polar growth, vesicle transport, and the expansion of hyphae in plant cells. Finally, transgenic rice (Oryza sativa) expressing RNA hairpins targeting MoFim1 exhibited improved resistance to M. oryzae infection, indicating that MoFim1 represents an excellent candidate for M. oryzae control. These results reveal the dynamics of actin assembly in M. oryzae during hyphal tip development and pathogenesis, and they suggest a mechanism in which MoFim1 organizes such actin networks.

Mesenchymal MACF1 Facilitates SMAD7 Nuclear Translocation to Drive Bone Formation.

Microtubule actin crosslinking factor 1 (MACF1) is a large crosslinker that contributes to cell integrity and cell differentiation. Recent studies show that MACF1 is involved in multiple cellular functions such as neuron development and epidermal migration, and is the molecular basis for many degenerative diseases. MACF1 is highly abundant in bones, especially in mesenchymal stem cells; however, its regulatory role is still less understood in bone formation and degenerative bone diseases. In this study, we found MACF1 expression in mesenchymal stem cells (MSCs) of osteoporotic bone specimens was significantly lower. By conditional gene targeting to delete the mesenchymal Macf1 gene in mice, we observed in MSCs decreased osteogenic differentiation capability. During early stage bone development, the MACF1 conditional knockout (cKO) mice exhibit significant ossification retardation in skull and hindlimb, and by adulthood, mesenchymal loss of MACF1 attenuated bone mass, bone microarchitecture, and bone formation capability significantly. Further, we showed that MACF1 interacts directly with SMAD family member 7 (SMAD7) and facilitates SMAD7 nuclear translocation to initiate downstream osteogenic pathways. Hopefully these findings will expand the biological scope of the MACF1 gene, and provide an experimental basis for targeting MACF1 in degenerative bone diseases such as osteoporosis.

Differential day-night expression of tight junction components in murine retinal pigment epithelium

Strong communication and interaction between the retinal pigment epithelium (RPE) and the photoreceptor (PR) cells is essential for vision. RPE cells are essential for supporting and maintaining PR cells by transporting nutrients, waste products and ions, and phagocytosing photoreceptor outer segments (POS). POS phagocytosis follows a circadian pattern, taking place in the morning in human, mice and other organisms. However, it remains unknown whether other RPE processes follow a daily rhythm. To study the daily rhythm of RPE cells, we isolated murine RPE cells at six different time points during a 24 h period, after which RNA was isolated and sequenced. Murine RPE flatmounts were isolated at four different time points to study daily rhythm in protein abundance and localisation. EnrichR pathway analysis resulted in 13 significantly-enriched KEGG pathways (p < 0.01) of which seven showed a large number of overlapping genes. Several genes were involved in intracellular trafficking, possibly playing a role in nutrient transport, POS phagocytosis or membrane protein trafficking, with different expression patterns during the day-night cycle. Other genes were involved in actin cytoskeleton building, remodelling and crosslinking and showed a high expression in the morning, suggesting actin cytoskeleton remodelling at this time point. Finally, tight junction proteins Cldn2 and Cldn4 showed a difference in RNA and protein expression and tight junction localisation over time. Our study suggests that several important processes in the RPE follow a day-night rhythm, including intracellular trafficking, and processes involving the actin cytoskeleton and tight junctions. The differential protein localisation of Cldn2 in the RPE during the day-night cycle suggest that Cldn2 may facilitate paracellular water and sodium transport during the day.

Filamin A inhibition reduces seizure activity in a mouse model of focal cortical malformations.

Epilepsy treatments for patients with mechanistic target of rapamycin (mTOR) disorders, such as tuberous sclerosis complex (TSC) or focal cortical dysplasia type II (FCDII), are urgently needed. In these patients, the presence of focal cortical malformations is associated with the occurrence of lifelong epilepsy, leading to severe neurological comorbidities. Here, we show that the expression of the actin cross-linking protein filamin A (FLNA) is increased in resected cortical tissue that is responsible for seizures in patients with FCDII and in mice modeling TSC and FCDII with mutations in phosphoinositide 3-kinase (PI3K)-ras homolog enriched in brain (Rheb) pathway genes. Normalizing FLNA expression in these mice through genetic knockdown limited cell misplacement and neuronal dysmorphogenesis, two hallmarks of focal cortical malformations. In addition, Flna knockdown reduced seizure frequency independently of mTOR signaling. Treating mice with a small molecule targeting FLNA, PTI-125, before the onset of seizures alleviated neuronal abnormalities and reduced seizure frequency compared to vehicle-treated mice. In addition, the treatment was also effective when injected after seizure onset in juvenile and adult mice. These data suggest that targeting FLNA with either short hairpin RNAs or the small molecule PTI-125 might be effective in reducing seizures in patients with TSC and FCDII bearing mutations in PI3K-Rheb pathway genes.

Dynamic Crosslinking of the Actin Cytoskeleton Governs Cell Mechanics

Cells precisely control their mechanical properties to organize and differentiate into tissues. The architecture and connectivity of cytoskeletal filaments changes in response to mechanical and biochemical cues, allowing the cell to rapidly tune its mechanics from highly-crosslinked, elastic networks to weakly-crosslinked viscous networks. While the role of actin crosslinking in controlling actin network mechanics is clear in purified actin networks, its mechanical role in the cytoplasm of living cells remains unknown. Here, we probe the frequency- dependent viscoelastic properties of the cytoplasm in living cells using multiharmonic excitation and in situ optical trap calibration. At previously unexplored long timescales, we observe that the cytoskeleton fluidizes. The mechanics are well-captured by a model in which actin filaments are dynamically connected by a single dominant crosslinker. A disease-causing point mutation (K255E) of the actin crosslinker α-actinin 4 (ACTN4) causes its binding kinetics to be insensitive to tension. Under normal conditions, the viscoelastic properties of wild type (WT) and K255E+/- cells are similar. However, when tension is reduced through myosin II inhibition, WT cells relax 3x faster to the fluid-like regime while K255E+/- cells are not affected. These results indicate that dynamic actin crosslinking enables the cytoplasm to flow at long timescales.

Bending of Actin Filaments into Rings by IQGAP Family of Proteins

Actin forming linear actin filaments builds up the acto-myosin cortex in cells providing mechanical stability, enabling vesicle transport, cell migration and cell division. This versality is achieved by interaction with many regulatory and actin binding proteins and the interplay with the plasma membrane to locally control actin filament length, stability and crosslinking to other filaments. Here, we report about a newly identified protein domain, Curly, within IQGAP family proteins that binds actin and causes bending of individual actin filaments into tight rings when anchored to a lipid membrane in vitro. Membrane anchored Curly induces reproducibly and efficiently F-actin ring formation of characteristic curvature (0.5-1 µm−1) and recognizes F-actin orientation. Interestingly, decoration of F-actin with the tropomyosin increases actin ring formation, while Fimbrin abrogates it. Addition of myosin II filaments results in further formation of actin rings by induced f-actin flow and, more importantly, in robust ring contraction. This constitutes, hence, a minimal (four components) contractile ring system. The discovery of Curly provides a new opportunity to address and control F-actin curvature in cells and in vitro and will open new approaches to generate synthetic cells capable of division and controlled shape changes.

Actin Controls the Dynamics and Microtubule Crosslinkers Tune Co-localization in Crosslinked Composite Actin-microtubule Networks

The cytoskeleton is a complex, active biomaterial system with the ability to control strain, localize objects in space and time, and self-organize and self-repair. Recent advances in composite networks of actin filaments and microtubules are better replicating cellular systems and promoting new avenues for biomaterials research in composite materials. Each type of filament comes with a host of accessory proteins and enzymes that can enable a wide array of activities. Here, we combine actin and microtubule filaments in the presence of increasing crosslinkers. For actin, we use permanent biotin-streptavidin crosslinkers to bundle and strengthen the actin network. For microtubules, we use the antiparallel crosslinker, MAP65. The presence of high concentrations of MAP65 causes actin to co-localize with microtubules. We find that the mobility of filaments in the network is dominated by the degree of crosslinking by actin filaments. The effect of microtubule crosslinking only affects filament mobility at intermediate actin crosslinking. This work demonstrates that actin filaments serve as the component that controls network dynamics, whereas microtubules have more control over network organization and localization.

Visualizing Dynamic Actin Crosslinking Processes Driven by the Actin Binding Protein Anillin

Anillin is an actin binding protein that can bundle actin filaments and associate with the plasma membrane in the ring-like actin network. Anillin can be divided into a membrane-bound domain and an actin binding domain (ABD). Although the ABD in anillin plays an important role in the dynamic ring transition, it is unclear how the ABD interacts with the actin ring structures during the contractile ring to midbody ring transition. In order to elucidate the underlying intermolecular interactions between the ABD and actin filaments, it is necessary to observe dynamic crosslinking processes. Here, we performed real-time imaging of actin filament crosslinking dynamics by the ABD of anillin using total internal reflection fluorescence microscopy (TIRFM) and high-speed atomic force microscopy (Hs-AFM) for single-molecule imaging. Both human full-length anillin and anillin N-terminal ABD constructs existed as monomers and showed relatively low binding affinity for actin filaments. Hs-AFM showed that anillin monomers crosslinked with actin filaments at a distance of 8 nm. The distance between the aligned crosslinked anillin molecules was approximately 37 nm, which corresponded to the half pitch of an actin filament. Furthermore, the polarity of these filaments was both parallel and antiparallel. Our direct and dynamic high-resolution visualization of the actin crosslinking process provides a powerful new approach to studying the interactions and dynamics of biomolecules.

Non-Ergodic Transport and Conformational Dynamics of DNA in Biomimetic Cytoskeleton Networks

Macromolecular crowding is an increasingly relevant biophysical phenomenon affecting gene expression, drug delivery, protein function, and intracellular transport. Of particular interest is the influence that cytoskeletal filaments have on the transport and conformational dynamics of large DNA molecules -- two properties critical for processes such as transfection, viral infection, and gene therapy. Here, we use single-molecule conformational tracking (SMCT) to elucidate the transport properties and conformational dynamics of linear and relaxed circular (ring) DNA in in vitro composite networks of actin and microtubules with variable types of crosslinking. Specifically, we investigate the impact of crosslinking actin to actin, microtubules to microtubules, and actin to microtubules. While both linear and ring DNA undergo anomalous subdiffusion in all networks, the transport properties are heavily influenced by DNA topology (linear vs ring). Linear DNA chains are compacted and display a single mode of subdiffusion, while ring DNA polymers are swollen and exhibit biphasic subdiffusion suggestive of transient threading by the biomimetic cytoskeleton. These results are bolstered by the non-Gaussian nature of displacement probabilities and the non-ergodic behavior of both DNAs, with the relaxed circular DNA becoming less ergodic than their linear counterparts at long times.