Actin-microtubule dynamic composite forms responsive active matter with memory

Active cytoskeletal materials in vitro demonstrate self-organising properties similar to those observed in their counterparts in cells. However, the search to emulate phenomena observed in the living matter has fallen short of producing a cytoskeletal network that would be structurally stable yet possessing adaptive plasticity. Here, we address this challenge by combining cytoskeletal polymers in a composite, where self-assembling microtubules and actin filaments collectively self-organise due to the activity of microtubules-percolating molecular motors. We demonstrate that microtubules spatially organise actin filaments that in turn guide microtubules. The two networks align in an ordered fashion using this feedback loop. In this composite, actin filaments can act as structural memory and, depending on the concentration of the components, microtubules either write this memory or get guided by it. The system is sensitive to external stimuli suggesting possible autoregulatory behaviour in changing mechanochemical environment. We thus establish artificial active actin-microtubule composite as a system demonstrating architectural stability and plasticity.

Structural mechanism for bi-directional actin crosslinking by T-plastin

To fulfill the cytoskeleton's diverse functions in cell mechanics and motility, actin networks with specialized architectures are built by crosslinking proteins, which bridge filaments to control micron-scale network geometry through nanoscale binding interactions via poorly defined structural mechanisms. Here, we introduce a machine-learning enabled cryo-EM pipeline for visualizing active crosslinkers, which we use to analyze human T-plastin, a member of the evolutionarily ancient plastin/fimbrin family of tandem calponin-homology domain (CHD) proteins. We define a sequential bundling mechanism which enables T-plastin to bridge filaments in both parallel and anti-parallel orientations. Our structural, biochemical, and cell biological data highlight inter-CHD linkers as key structural elements underlying flexible but stable crosslinking which are likely to be disrupted by mutations causing hereditary bone diseases. Beyond revealing how plastins are evolutionary optimized to crosslink dense actin networks with mixed polarity, our cryo-EM workflow will broadly enable analysis of the structural mechanisms underlying cytoskeletal network construction.

Role of the cytoskeleton in steroidogenesis

: Steroidogenesis in the adrenal cortex or gonads is a complicated process, modulated by various elements either at the tissue or molecular level. The substrate—cholesterol is first delivered to the outer membrane of mitochondria, undergoing a series of enzymatic reactions along with the material exchange between the mitochondria and the ER (endoplasmic reticulum) and ultimately yield various steroids: aldosterone, cortisol, testosterone and estrone. Several valves are set to adjust the amount of production to the needs. e.g. StAR(steroidogenic acute regulator) is in charge of the rate-limiting step—traffic of cholesterol from outer membrane to inner membrane of mitochondria. And the “needs” is partly reflected by trophic signals like ACTH、LH and downstream pathways-- intracellular cAMP pathway, which represents the endocrinal regulation of steroid synthesis, too. The coordinated activities of these related factors are all associated with another crucial cellular constituent—the cytoskeleton, which plays a crucial role in the cellular architecture and substrate trafficking. Though considerable studies have been performed regarding steroid synthesis, details about the upstream signaling pathways and mechanisms of the regulation by cytoskeleton network still remain unclear. The metabolism and interplays of the pivotal cellular organelles with cytoskeleton are worth exploring as well. In this review, we summarize research of different time span, describing the roles of specific cytoskeleton elements in steroidogenesis and related signaling pathways involved in the steroid synthesis. In addition, we discussed the inner cytoskeletal network involved in steroidogenic processes such as mitochondrial movement, organelle interactions and cholesterol trafficking.

New perspectives on cytoskeletal dysregulation and mitochondrial mislocalization in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective, early degeneration of motor neurons in the brain and spinal cord. Motor neurons have long axonal projections, which rely on the integrity of neuronal cytoskeleton and mitochondria to regulate energy requirements for maintaining axonal stability, anterograde and retrograde transport, and signaling between neurons. The formation of protein aggregates which contain cytoskeletal proteins, and mitochondrial dysfunction both have devastating effects on the function of neurons and are shared pathological features across several neurodegenerative conditions, including ALS, Alzheimer's disease, Parkinson's disease, Huntington’s disease and Charcot-Marie-Tooth disease. Furthermore, it is becoming increasingly clear that cytoskeletal integrity and mitochondrial function are intricately linked. Therefore, dysregulations of the cytoskeletal network and mitochondrial homeostasis and localization, may be common pathways in the initial steps of neurodegeneration. Here we review and discuss known contributors, including variants in genetic loci and aberrant protein activities, which modify cytoskeletal integrity, axonal transport and mitochondrial localization in ALS and have overlapping features with other neurodegenerative diseases. Additionally, we explore some emerging pathways that may contribute to this disruption in ALS.

Cell responses to simulated microgravity and hydrodynamic stress can be distinguished by using comparative transcriptomic analysis

The human immune system is compromised in microgravity (MG) conditions during an orbital flight and upon return to Earth. T cells are critical for the immune response and execute their functions via actin mediated immune cell-cell interactions that could be disturbed by MG conditions. Here, we have applied two rotational platforms to simulate MG conditions: fast rotating clinostat (CL) and random positioning machine (RPM) followed by global T cell transcriptome analysis using RNA sequencing. We demonstrate that the T cell transcriptome profile in response to simulated MG treatment was clearly distinguishable from the T cell transcriptome response to hydrodynamic stress (HS) induced by shear forces upon cell movement in cultural medium. Gene expression profiling of genes related to or involved in actin cytoskeleton networks using RT-qPCR confirmed two sets of differentially regulated genes in the T cell response to MG or to HS. Several key genes potentially involved in T cell gravisensing (Fam163b, Dnph1, Trim34, Upk-1b) were identified. A number of candidate biomarker genes of the response to MG (VAV1, VAV2, VAV3, and NFATC2) and of the response to HS (ITGAL, ITGB1, ITGB2, RAC1 and RAC2) could be used to distinguish between these processes on the gene transcription level. Together, MG induces changes in the overall transcriptome of T cells leading to specific shifts in expression of cytoskeletal network genes.

Upregulation of Cx43 Expression Under Stretch Condition is Mediated by TGF Beta1 and Cytoskeletal Network

Many physiological and pathophysiological processes in cells or tissues are involving mechanical stretch, which is inducing gap junction gene expression and cytokine TGF beta changes. However, the underlying mechanisms of gap junction gene expression changes remain unknown. Here, we showed that the expression of Cx43 at mRNA and protein level in Human umbilical-vein endothelial cells (HUVECs) is significantly increased after 24 h stretch stimulation, and TGF beta1, but not TGF beta2 expression is also upregulated. Administration of TGF betal into HUVECs without stretch also induced upregulation of Cx43 mRNA and protein expression. While simultaneously administration of TGF beta1 with SB431542, a specific inhibitor of TGF beta1 receptor, blocked the Cx43 protein upregulation by TGF beta1. Further, the increase of Cx43 protein expression under stretch condition can be partially blocked by SB431542; moreover, it also can be partially blocked by simultaneously administration of anti-TGF beta1 monoclonal neutralization antibody. Importantly, the stretch induced upregulation of Cx43 can be blocked by administration of actin and microtubule inhibitors, while NEDD4, a key element in mediating Cx43 protein ubiquitination and degradation, is not changed under stretch condition. Therefore, we conclude that upregulation of Cx43 expression under 24 h stretch condition is mediated by TGF beta1 via TGF beta1 receptor signaling pathway, and it also involves the actin and microtubule cytoskeletal network.

Electrophysiological analysis of healthy and dystrophic 3D bioengineered skeletal muscle tissues

Recently, methods for creating three-dimensional (3D) human skeletal muscle tissues from myogenic cell lines have been reported. Bioengineered muscle tissues are contractile and respond to electrical and chemical stimulation. In this study we provide an electrophysiological analysis of healthy and dystrophic 3D bioengineered skeletal muscle tissues. We focus on Duchenne muscular dystrophy (DMD), a fatal muscle disorder involving the skeletal muscle system. The dystrophin gene, which when mutated causes DMD, encodes for the Dystrophin protein, which anchors the cytoskeletal network inside of a muscle cell to the extracellular matrix outside the cell. Here, we enlist a 3D in vitro model of DMD muscle tissue, to evaluate an understudied aspect of DMD, muscle cell electrical properties uncoupled from presynaptic neural inputs. Our data shows that electrophysiological aspects of DMD are replicated in the 3D bioengineered skeletal muscle tissue model. Furthermore, we test a block co-polymer, poloxamer 188, and demonstrate capacity for improving the membrane potential in DMD muscle. Therefore, this study serves as the baseline for a new in vitro method to examine potential therapies directed at muscular disorders.

They Might Cut It—Lysosomes and Autophagy in Mitotic Progression

The division of one cell into two looks so easy, as if it happens without any control at all. Mitosis, the hallmark of mammalian life is, however, tightly regulated from the early onset to the very last phase. Despite the tight control, errors in mitotic division occur frequently and they may result in various chromosomal instabilities and malignancies. The flow of events during mitotic progression where the chromosomes condensate and rearrange with the help of the cytoskeletal network has been described in great detail. Plasma membrane dynamics and endocytic vesicle movement upon deadhesion and reattachment of dividing cells are also demonstrated to be functionally important for the mitotic integrity. Other cytoplasmic organelles, such as autophagosomes and lysosomes, have until recently been considered merely as passive bystanders in this process. Accordingly, at the onset of nuclear envelope breakdown in prometaphase, the number of autophagic structures and lysosomes is reduced and the bulk autophagic machinery is suppressed for the duration of mitosis. This is believed to ensure that the exposed nuclear components are not unintentionally delivered to autophagic degradation. With the evolving technologies that allow the detection of subtle alterations in cytoplasmic organelles, our understanding of the small-scale regulation of intracellular organelles has deepened rapidly and we discuss here recent discoveries revealing unexpected roles for autophagy and lysosomes in the preservation of genomic integrity during mitosis.

An Energetic Approach to Modeling Cytoskeletal Architecture in Maturing Cardiomyocytes

Through a variety of mechanisms, a healthy heart is able to regulate its structure and dynamics across multiple length scales. Disruption of these mechanisms can have a cascad- ing effect, resulting in severe structural and/or functional changes that permeate across different length scales. Due to this hierarchical structure, there is interest in understand- ing how the components at the various scales coordinate and influence each other. However, much is unknown regarding how myofibril bundles are organized within a densely packed cell and the influence of the subcellular components on the architecture that is formed. To elucidate potential factors influencing cytoskeletal development, we proposed a compu- tational model that integrated interactions at both the cel- lular and subcelluar scale to predict the location of indi- vidual myofibril bundles that contributed to the formation of an energetically favorable cytoskeletal network. Our model was tested and validated using experimental metrics derived from analyzing single cell cardiomyocytes. We demonstrated that our model-generated networks were capable of repro- ducing the variation observed in experimental cells at different length scales as a result of the stochasticity inher- ent in the different interaction between the various cellu- lar components. Additionally, we showed that incorporat- ing length-scale parameters resulted in physical constraints that directed cytoskeletal architecture towards a structurally consistent motif. Understanding the mechanisms guiding the formation and organization of the cytoskeleton in individual cardiomyocytes can aid tissue engineers towards developing functional cardiac tissue.

Shaping liposomes by cell-free expressed bacterial microtubules

Genetic control over a cytoskeletal network inside lipid vesicles offers a potential route to controlled shape changes and DNA segregation in synthetic cell biology. Bacterial microtubules (bMTs) are protein filaments found in bacteria of the genus Prosthecobacter. They are formed by the tubulins BtubA and BtubB which polymerize in the presence of GTP. Here, we show that the tubulins BtubA/B can be functionally expressed from DNA templates in a reconstituted transcription-translation system, thus providing a cytosol-like environment to study their biochemical and biophysical properties. We found that bMTs spontaneously interact with lipid membranes and display treadmilling. When compartmentalized inside liposomes, de novo synthesized BtubA/B tubulins self-organize into cytoskeletal structures of different morphologies. Moreover, bMTs can exert a pushing force on the membrane and deform liposomes, a phenomenon that can be reversed by light-activated disassembly of the filaments. Our work establishes bMTs as a new building block in synthetic biology. In the context of creating a synthetic cell, bMTs could help shape the lipid compartment, establish polarity or directional transport, and assist the division machinery.