Cytoskeletal Scaffold Research Articles

ANGI-13. LONG NON-CODING RNALINC03045 REGULATES GLIOBLASTOMA INVASION VIAWASF3

Abstract Glioblastoma (GBM) is an aggressive, heterogeneous tumor characterized by extensive infiltration of tumor cells within the surrounding brain parenchyma. Invasive tumor cells that evade surgical resection, chemoradiation and immune responses underlie GBM recurrence, necessitating the evaluation of factors that govern them. A recently completed unbiased lncRNA CRISPRi screen done by our lab identified LINC03045 as the top potential regulator of GBM invasion in Matrigel assays. Since enhanced LINC03045 expression was also found to be significantly associated with glioma tumor grade and patient survival (p<0.0001), here we characterized potential mechanisms by which it regulates GBM invasion. Mediators of lncRNA effect were identified via differential expression analysis in LINC03045 knockdown glioma cells and TCGA glioma datasets, and assessed for tumor invasion in 2D and 3D assays using knockdown and rescue experiments. Differential expression analysis of protein-coding genes associated with reduced LINC03045 expression in vitro and in GBM TCGA RNAseq data revealed high positive correlation with WASF3 (p=0.016), a regulator of JAK-STAT signaling and cytoskeletal scaffolding. siRNA-mediated WASF3 knockdown was associated with significant decrease in invasion in 2D-Matrigel assays in U87 (16.4%, p<0.001), U251(27.3%, p<0.001) and patient-derived USC02 (25.7%, p<0.001) glioma cells. Additionally, 3D invasion assays showed reduced invasion of WASF3KD U87 gliomaspheres (41.1%, p=0.011) into the surrounding matrix. Finally, WASF3 overexpression in USC02s demonstrated rescue of invasive capacity lost with LINC03045 knockdown (p<0.01). Taken together, RNA-seq analysis of downstream effectors of LINC03045, coupled with tumor invasion studies establish downstream mediator WASF3 as a novel regulator of GBM invasive potential.

Mechanosensory entities and functionality of endothelial cells.

The endothelial cells of the blood circulation are exposed to hemodynamic forces, such as cyclic strain, hydrostatic forces, and shear stress caused by the blood fluid's frictional force. Endothelial cells perceive mechanical forces via mechanosensors and thus elicit physiological reactions such as alterations in vessel width. The mechanosensors considered comprise ion channels, structures linked to the plasma membrane, cytoskeletal spectrin scaffold, mechanoreceptors, and junctional proteins. This review focuses on endothelial mechanosensors and how they alter the vascular functions of endothelial cells. The current state of knowledge on the dysregulation of endothelial mechanosensitivity in disease is briefly presented. The interplay in mechanical perception between endothelial cells and vascular smooth muscle cells is briefly outlined. Finally, future research avenues are highlighted, which are necessary to overcome existing limitations.

Biallelic EPB41L3 variants underlie a developmental disorder with seizures and myelination defects.

Erythrocyte Membrane Protein Band 4.1 Like 3 (EPB41L3: NM_012307.5), also known as DAL-1, encodes the ubiquitously expressed, neuronally enriched 4.1B protein, part of the 4.1 superfamily of membrane-cytoskeleton adaptors. 4.1B plays key roles in cell spreading, migration, and cytoskeletal scaffolding that support oligodendrocyte axon adhesions essential for proper myelination. We herein describe six individuals from five unrelated families with global developmental delay, intellectual disability, seizures, hypotonia, neuroregression, and delayed myelination. Exome sequencing identified biallelic variants in EPB41L3 in all affected individuals: two nonsense (c.466C>T, p.(R156*); c.2776C>T, p.(R926*)) and three frameshift (c.666delT, p.(F222Lfs*46); c.2289dupC, p.(V764Rfs*19); c.948_949delTG, p.(A317Kfs*33)). Quantitative-real time PCR and Western blot analysis in human fibroblasts harbouring EPB41L3:c.666delT, p.(F222Lfs*46) indicate ablation of EPB41L3 mRNA and 4.1B protein expression. Inhibition of the nonsense mediated decay (NMD) pathway led to an upregulation of EPB41L3:c.666delT transcripts, supporting NMD as a pathogenic mechanism. Epb41l3-deficient mouse oligodendroglia cells showed significant reduction in mRNA expression of key myelin genes, reduced branching, and increased apoptosis. Our report provides the first clinical description of an autosomal recessive disorder associated with variants in EPB41L3, which we refer to as EPB41L3-associated developmental disorder (EADD). Moreover, our functional studies substantiate the pathogenicity of EPB41L3 hypothesized loss-of-function variants.

Building on-chip cytoskeletal circuits via branched microtubule networks

Controllable platforms to engineer robust cytoskeletal scaffolds have the potential to create novel on-chip nanotechnologies. Inspired by axons, we combined the branching microtubule (MT) nucleation pathway with microfabrication to develop "cytoskeletal circuits." This active matter platform allows control over the adaptive self-organization of uniformly polarized MT arrays via geometric features of microstructures designed within a microfluidic confinement. We build and characterize basic elements, including turns and divisions, as well as complex regulatory elements, such as biased division and MT diodes, to construct various MT architectures on a chip. Our platform could be used in diverse applications, ranging from efficient on-chip molecular transport to mechanical nano-actuators. Further, cytoskeletal circuits can serve as a tool to study how the physical environment contributes to MT architecture in living cells.

Novel pathogenic WHRN variant causing hearing loss in a moroccan family.

The most prevalent sensory disease in humans is deafness. A variety of genes have been linked to hearing loss, which can either be isolated (non-syndromic) or associated with lesions in other organs (syndromic). It has been discovered that WHRN variants are responsible for non-syndromic hearing loss and Usher syndrome type II. Exome sequencing in a consanguineous Moroccan patient with severe hearing loss identified a single homozygous mutation c.619G > T; p.Ala207Ser in WHRN, encoding a cytoskeletal scaffold protein that binds membrane protein complexes to the cytoskeleton in ocular photoreceptors and ear hair cell stereocilia. Bioinformatics methods and molecular dynamic modeling were able to predict the pathogenic implications of this variation. We used whole exome sequencing to find a homozygous WHRN gene variant in a Moroccan family. Numerous bioinformatics methods predict that this modification might result in a change in the WHRN protein's structure.

Evidence of two differentially regulated elongasomes in Salmonella

Cell shape is genetically inherited by all forms of life. Some unicellular microbes increase niche adaptation altering shape whereas most show invariant morphology. A universal system of peptidoglycan synthases guided by cytoskeletal scaffolds defines bacterial shape. In rod-shaped bacteria, this system consists of two supramolecular complexes, the elongasome and divisome, which insert cell wall material along major and minor axes. Microbes with invariant shape are thought to use a single morphogenetic system irrespective of the occupied niche. Here, we provide evidence for two elongasomes that generate (rod) shape in the same bacterium. This phenomenon was unveiled in Salmonella, a pathogen that switches between extra- and intracellular lifestyles. The two elongasomes can be purified independently, respond to different environmental cues, and are directed by distinct peptidoglycan synthases: the canonical PBP2 and the pathogen-specific homologue PBP2SAL. The PBP2-elongasome responds to neutral pH whereas that directed by PBP2SAL assembles in acidic conditions. Moreover, the PBP2SAL-elongasome moves at a lower speed. Besides Salmonella, other human, animal, and plant pathogens encode alternative PBPs with predicted morphogenetic functions. Therefore, contrasting the view of morphological plasticity facilitating niche adaptation, some pathogens may have acquired alternative systems to preserve their shape in the host.

Nodes of Ranvier in health and disease.

Action potential propagation along myelinated axons depends on the geometry of the myelin unit and the division of the underlying axon to specialized domains. The latter include the nodes of Ranvier (NOR), the paranodal junction (PNJ) flanking the nodes, and the adjacent juxtaparanodal region that is located below the compact myelin of the internode. Each of these domains contains a unique composition of axoglial adhesion molecules (CAMs) and cytoskeletal scaffolding proteins, which together direct the placement of specific ion channels at the nodal and juxtaparanodal axolemma. In the last decade it has become increasingly clear that antibodies to some of these axoglial CAMs cause immune-mediated neuropathies. In the current review we detail the molecular composition of the NOR and adjacent membrane domains, describe the function of different CAM complexes that mediate axon-glia interactions along the myelin unit, and discuss their involvement and the underlying mechanisms taking place in peripheral nerve pathologies. This growing group of pathologies represent a new type of neuropathies termed "nodopathies" or "paranodopathies" that are characterized by unique clinical and molecular features which together reflect the mechanisms underlying the molecular assembly and maintenance of this specialized membrane domain.

βII-Spectrin Is Required for Synaptic Positioning during Retinal Development.

Neural circuit assembly is a multistep process where synaptic partners are often born at distinct developmental stages, and yet they must find each other and form precise synaptic connections with one another. This developmental process often relies on late-born neurons extending their processes to the appropriate layer to find and make synaptic connections to their early-born targets. The molecular mechanism responsible for the integration of late-born neurons into an emerging neural circuit remains unclear. Here, we uncovered a new role for the cytoskeletal protein βII-spectrin in properly positioning presynaptic and postsynaptic neurons to the developing synaptic layer. Loss of βII-spectrin disrupts retinal lamination, leads to synaptic connectivity defects, and results in impaired visual function in both male and female mice. Together, these findings highlight a new function of βII-spectrin in assembling neural circuits in the mouse outer retina.SIGNIFICANCE STATEMENT Neurons that assemble into a functional circuit are often integrated at different developmental time points. However, the molecular mechanism that guides the precise positioning of neuronal processes to the correct layer for synapse formation is relatively unknown. Here, we show a new role for the cytoskeletal scaffolding protein, βII-spectrin in the developing retina. βII-spectrin is required to position presynaptic and postsynaptic neurons to the nascent synaptic layer in the mouse outer retina. Loss of βII-spectrin disrupts positioning of neuronal processes, alters synaptic connectivity, and impairs visual function.

Bacterial crystalline cellulose secretion via a supramolecular BcsHD scaffold.

Cellulose, the most abundant biopolymer on Earth, is not only the predominant constituent of plants but also a key extracellular polysaccharide in the biofilms of many bacterial species. Depending on the producers, chemical modifications, and three-dimensional assemblies, bacterial cellulose (BC) can present diverse degrees of crystallinity. Highly ordered, or crystalline, cellulose presents great economical relevance due to its ever-growing number of biotechnological applications. Even if some acetic acid bacteria have long been identified as BC superproducers, the molecular mechanisms determining the secretion of crystalline versus amorphous cellulose remain largely unknown. Here, we present structural and mechanistic insights into the role of the accessory subunits BcsH (CcpAx) and BcsD (CesD) that determine crystalline BC secretion in the Gluconacetobacter lineage. We show that oligomeric BcsH drives the assembly of BcsD into a supramolecular cytoskeletal scaffold that likely stabilizes the cellulose-extruding synthase nanoarrays through an unexpected inside-out mechanism for secretion system assembly.

Axonal architecture of the mouse inner retina revealed by second harmonic generation.

We describe a novel method for visualizing the network of axons in the unlabeled fresh wholemount retina. The intrinsic radiation of second harmonic generation (SHG) was utilized to visualize single axons of all major retinal neurons, i.e., photoreceptors, horizontal cells, bipolar cells, amacrine cells, and the retinal ganglion cells. The cell types of SHG+ axons were determined using transgenic GFP/YFP mice. New findings were obtained with retinal SHG imaging: Müller cells do not maintain uniformly polarized microtubules in the processes; SHG+ axons of bipolar cells terminate in the inner plexiform layer (IPL) in a subtype-specific manner; a subset of amacrine cells, presumably the axon-bearing types, emits SHG; and the axon-like neurites of amacrine cells provide a cytoskeletal scaffolding for the IPL stratification. To demonstrate the utility, retinal SHG imaging was applied to testing whether the inner retina is preserved in glaucoma, using DBA/2 mice as a model of glaucoma and DBA/2-Gpnmb+ as the nonglaucomatous control. It was found that the morphology of the inner retina was largely intact in glaucoma and the presynaptic compartments to the retinal ganglion cells were uncompromised. It proves retinal SHG imaging as a promising technology for studying the physiological and diseased retinas in 3D.

1367-P: A Submembrane Cytoskeleton-Dependent Mechanism Modulates Mitochondrial Homeostasis and Skeletal Muscle Bioenergetics

Human variants in the ubiquitously expressed cytoskeletal scaffolding protein Ankyrin B (AnkB) , which is enriched in skeletal muscle (SKM) , have been identified as risk factors for diabetes and obesity. Mice harboring pathogenic variants exhibit reductions in levels of the predominant 220-kDa AnkB isoform in several tissues, including SKM and impaired systemic glucose regulation. To understand the specific metabolic role of AnkB in SKM, we developed a conditional knock-out mouse in which 220-kDa AnkB is knocked out only in SKM (SKM-AnkB-KO) . SKM-AnkB-KO mice display glucose mishandling that persists with age, as well as decreases in activity and exercise capacity, measured by indirect calorimetry and voluntary running wheel analysis. Histological evaluations of aged SKM-AnkB-KO mice showed general disorganization of muscle fibers and the presence of centralized nuclei. This is accompanied by increases in lean muscle mass and in the cross-sectional area of mitochondria-rich soleus muscle, suggesting the development of muscle hypertrophy over time. Electron microscopy analysis revealed the presence of hyperfused mitochondria in SKM-AnkB-KO mice. This morphological change was concomitant with up-regulation of transcripts in pathways associated with energetic stress, including AMPK signaling, redox signaling and mTOR signaling, assessed by RNA-seq, and with alterations in mitochondrial respiration in isolated muscle fibers. Interestingly, SKM-AnkB-KO mice mi SKM-AnkB-KO mice also exhibit increases in protein levels of electron transport chain proteins. Through proteomics and biochemical analysis, we uncovered that AnkB directly associates with several mitochondrial proteins and co-fractionates with mitochondrial in SKM. Together, these data suggest a direct role of AnkB in SKM mitochondrial metabolism that is distinct from its previously characterized role in insulin signaling in other tissues and cytoskeletal organization in SKM. Disclosure K.Voos: n/a. J.Tzeng: None. H.Choi: None. T.Pharr: None. D.Lorenzo: None. Funding American Diabetes Association; National Institutes of Health (1T32GM119999-01A1)

245-LB: An Ankyrin-B–Mediated Protein Chaperone Mechanism Modulates Pancreatic ß-Cell Susceptibility to Proteotoxicity-Induced ER Stress and Apoptosis

Impaired insulin secretion contributes to the development and progression of diabetes. β-cells initially cope with the genetic, environmental, and diabetogenic stresses that diminish insulin secretion or the peripheral response to it by increasing insulin production. However, sustained increases in the insulin precursor proinsulin (PI) place additional demand on the secretory and protein quality control (PQC) machineries of β-cells, triggering endoplasmic reticulum (ER) and oxidative stress, mitochondrial dysfunction, and β-cell death. Thus, understanding the mechanisms that regulate β-cell responses to increased insulin secretory requirements is critical to prevent β-cell failure. We reported that the cytoskeletal scaffolding protein ankyrin-B (AnkB) regulates insulin secretion by maintaining normal levels of the inositol-1,4,5-triphosphate receptor (IP3R) at the ER and intracellular calcium in β-cells. IP3R levels and insulin secretion were impaired in knock-in mice carrying the p.R1788W AnkB (AnkB-RW) variant associated with type 2 diabetes (T2D) in Caucasians and Hispanics. Here, we show that selective loss of AnkB or expression of the T2D AnkB-RW variant lead to reduced insulin secretion and accumulation of PI and islet specific amyloid polypeptide in β-cells. These changes are accompanied by enhanced induction of the maladaptive unfolded protein response and apoptosis. Interestingly, the T2D AnkB-RW variant lies within the binding site for the protein chaperone Dnajb1 (Hsp40) . Our results indicate that the Dnajb1-Hspa1a complex, which recognizes and delivers protein clients to Hsp70 chaperones to promote proper folding, refolding, or degradation, is downregulated in β-cells from β-cell-specific AnkB knockout or AnkB-RW mice. Increased proteotoxicity and reduced insulin secretion were also observed upon knockdown of Dnajb1 in rat INS1 832/13 cells. Together, our findings suggest that AnkB and its integration into the Dnajb1-Hspa1a pathway modulates PQC and promotes β-cell survival under diabetogenic conditions. Disclosure J. Tzeng: None. H. Hohmeier: None. D. Lorenzo: None.

Autism-associated ANK2 regulates embryonic neurodevelopment

Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by altered social communication, restricted interests, and stereotypic behaviors. Although the molecular and cellular pathogeneses of ASD remain elusive, impaired neural stem cell differentiation and neuronal migration during cortical development are suggested to be critically involved in ASD. ANK2, which encodes for a cytoskeletal scaffolding protein involved in recruiting membrane proteins into specialized membrane domains, has been identified as a high-confidence ASD risk gene. However, the role of ANK2 in early neural development remains unclear. In this study, we analyzed the role of ANK2 in the cerebral cortex of developing mouse using in utero electroporation. We provide evidence suggesting that ANK2 regulates neural stem cell differentiation and neuronal migration in the embryonic cerebral cortex, where Ank2 is highly expressed. We also demonstrated that Ank2 knockdown alters the expression of genes involved in neural development. Taken together, these results support the view that ANK2 haploinsufficiency in patients may impair neural development, resulting in an increased risk of ASD. Our study findings provide new insights into the molecular and cellular pathogenesis of ASD, given that among high-confidence ASD genes, ANK2 is rare in that it encodes for a scaffolding protein for the membrane protein complex required for neuronal functions.

Reciprocal EGFR signaling in the anchor cell ensures precise inter-organ connection during Caenorhabditis elegans vulval morphogenesis.

ABSTRACTDuring Caenorhabditis elegans vulval development, the uterine anchor cell (AC) first secretes an epidermal growth factor (EGF) to specify the vulval cell fates and then invades the underlying vulval epithelium. By doing so, the AC establishes direct contact with the invaginating primary vulF cells and attaches the developing uterus to the vulva. The signals involved and the exact sequence of events joining these two organs are not fully understood. Using a conditional let-23 EGF receptor (EGFR) allele along with novel microfluidic short- and long-term imaging methods, we discovered a specific function of the EGFR in the AC during vulval lumen morphogenesis. Tissue-specific inactivation of let-23 in the AC resulted in imprecise alignment of the AC with the primary vulval cells, delayed AC invasion and disorganized adherens junctions at the contact site forming between the AC and the dorsal vulF toroid. We propose that EGFR signaling, activated by a reciprocal EGF cue from the primary vulval cells, positions the AC at the vulval midline, guides it during invasion and assembles a cytoskeletal scaffold organizing the adherens junctions that connect the developing uterus to the dorsal vulF toroid. Thus, EGFR signaling in the AC ensures the precise alignment of the two developing organs.

Cross Talk between ARF1 and RhoA Coordinates the Formation of Cytoskeletal Scaffolds during Chlamydia Infection.

ABSTRACTChlamydia trachomatis is an obligate intracellular bacterium that has developed sophisticated mechanisms to survive inside its infectious compartment, the inclusion. Notably, Chlamydia weaves an extensive network of microtubules (MTs) and actin filaments to enable interactions with host organelles and enhance its stability. Despite the global health and economic burden caused by this sexually transmitted pathogen, little is known about how actin and MT scaffolds are integrated into an increasingly complex virulence system. Previously, we established that the chlamydial effector InaC interacts with ARF1 to stabilize MTs. We now demonstrate that InaC regulates RhoA to control actin scaffolds. InaC relies on cross talk between ARF1 and RhoA to coordinate MTs and actin, where the presence of RhoA downregulates stable MT scaffolds and ARF1 activation inhibits actin scaffolds. Understanding how Chlamydia hijacks complex networks will help elucidate how this clinically significant pathogen parasitizes its host and reveal novel cellular signaling pathways.

Plectin in Skin Fragility Disorders.

Plectin is a multi-faceted, 500 kDa-large protein, which due to its expression in different isoforms and distinct organs acts diversely as a cytoskeletal crosslinker and signaling scaffold. It functions as a mediator of keratinocyte mechanical stability in the skin, primarily through linking intermediate filaments to hemidesmosomes. Skin fragility may occur through the presence of mutations in the gene encoding for plectin, PLEC, or through the presence of autoantibodies against the molecule. Below, we review the cutaneous manifestations of plectinopathies as well as their systemic involvement in specific disease subtypes. We summarize the known roles of plectin in keratinocytes and fibroblasts and provide an outlook on future perspectives for plectin-associated skin disorders.

Photoreceptor Compartment-Specific TULP1 Interactomes

Photoreceptors are highly compartmentalized cells with large amounts of proteins synthesized in the inner segment (IS) and transported to the outer segment (OS) and synaptic terminal. Tulp1 is a photoreceptor-specific protein localized to the IS and synapse. In the absence of Tulp1, several OS-specific proteins are mislocalized and synaptic vesicle recycling is impaired. To better understand the involvement of Tulp1 in protein trafficking, our approach in the current study was to physically isolate Tulp1-containing photoreceptor compartments by serial tangential sectioning of retinas and to identify compartment-specific Tulp1 binding partners by immunoprecipitation followed by liquid chromatography tandem mass spectrometry. Our results indicate that Tulp1 has two distinct interactomes. We report the identification of: (1) an IS-specific interaction between Tulp1 and the motor protein Kinesin family member 3a (Kif3a), (2) a synaptic-specific interaction between Tulp1 and the scaffold protein Ribeye, and (3) an interaction between Tulp1 and the cytoskeletal protein microtubule-associated protein 1B (MAP1B) in both compartments. Immunolocalization studies in the wild-type retina indicate that Tulp1 and its binding partners co-localize to their respective compartments. Our observations are compatible with Tulp1 functioning in protein trafficking in multiple photoreceptor compartments, likely as an adapter molecule linking vesicles to molecular motors and the cytoskeletal scaffold.

SPTAN1 Expression Predicts Treatment and Survival Outcomes in Colorectal Cancer.

Simple SummaryColorectal cancer (CRC) is a common and deadly form of cancer. Non-erythroid spectrin αII (SPTAN1), a protein of the cytoskeleton, is thought to be involved in CRC development and progression. In this study, we explore whether measuring SPTAN1 levels in resected CRC specimens might help to predict patient survival outcomes and response to chemotherapy. Indeed, we find that higher SPTAN1 protein and mRNA levels in CRC specimens associate with longer patient survival times. Using cell culture experiments, we then show that cells with lower SPTAN1 levels are less susceptible to FOLFOX chemotherapy, a standard treatment regimen for patients with CRC. Overall, our study underscores the importance of cytoskeletal proteins in shaping tumour biology and treatment responses and nominates SPTAN1 as a biomarker to improve patient stratification and refine therapeutic decisions in CRC.Colorectal cancer (CRC) is a leading cause of cancer-related morbidity and mortality. In a cohort of 189 patients with CRC, we recently showed that expression of the cytoskeletal scaffolding protein non-erythroid spectrin αII (SPTAN1) was lower in advanced metastatic tumours. The aim of the present study was to clarify the association of intratumoural SPTAN1 expression levels with treatment and survival outcomes in patients with CRC. The analysis was based on histologic assessment of SPTAN1 protein levels in our own CRC cohort, and transcriptome data of 573 CRC cases from The Cancer Genome Atlas (TCGA). We first establish that high intratumoural levels of SPTAN1 protein and mRNA associate with favourable survival outcomes in patients with CRC. Next, a response prediction signature applied to the TCGA data reveals a possible link between high SPTAN1 transcript levels and improved patient responses to FOLFOX chemotherapy. Complementary in vitro experiments confirm that SPTAN1 knockdown strains of the colon cancer cell lines HT-29, HCT116 mlh1-2 and Caco-2 are less responsive to FOLFOX chemotherapy compared with SPTAN1-proficient control strains. Taken together, we identify SPTAN1 as a novel prognostic biomarker in CRC and show that SPTAN1 expression levels may predict patient responses to chemotherapy. These investigations illustrate how an affordable, histology-based diagnostic test could directly impact therapeutic decision-making at the bedside.

Mitochondrial dynamics in health and disease.

In animals, mitochondria are mainly organised into an interconnected tubular network extending across the cell along a cytoskeletal scaffold. Mitochondrial fission and fusion, as well as distribution along cytoskeletal tracks, are counterbalancing mechanisms acting in concert to maintain a mitochondrial network tuned to cellular function. Balanced mitochondrial dynamics permits quality control of the network including biogenesis and turnover, and distribution of mitochondrial DNA, and is linked to metabolic status. Cellular and organismal health relies on a delicate balance between fission and fusion, and large rearrangements in the mitochondrial network can be seen in response to cellular insults and disease. Indeed, dysfunction in the major components of the fission and fusion machineries including dynamin-related protein 1 (DRP1), mitofusins 1 and 2 (MFN1, MFN2) and optic atrophy protein 1 (OPA1) and ensuing imbalance of mitochondrial dynamics can lead to neurodegenerative disease. Altered mitochondrial dynamics is also seen in more common diseases. In this review, the machinery involved in mitochondrial dynamics and their dysfunction in disease will be discussed.

Impact of type-1 collagen hydrogel density on integrin-linked morphogenic response of SH-SY5Y neuronal cells.

Cellular metabolism and behaviour is closely linked to cytoskeletal tension and scaffold mechanics. In the developing nervous system functional connectivity is controlled by the interplay between chemical and mechanical cues that initiate programs of cell behaviour. Replication of functional connectivity in neuronal populations in vitro has proven a technical challenge due to the absence of many systems of biomechanical regulation that control directional outgrowth in vivo. Here, a 3D culture system is explored by dilution of a type I collagen hydrogel to produce variation in gel stiffness. Hydrogel scaffold remodelling was found to be linked to gel collagen concentration, with a greater degree of gel contraction occurring at lower concentrations. Gel mechanics were found to evolve over the culture period according to collagen concentration. Less concentrated gels reduced in stiffness, whilst a biphasic pattern of increasing and then decreasing stiffness was observed at higher concentrations. Analysis of these cultures by PCR revealed a program of shifting integrin expression and highly variable activity in key morphogenic signal pathways, such as mitogen-associated protein kinase, indicating genetic impact of biomaterial interactions via mechano-regulation. Gel contraction at lower concentrations was also found to be accompanied by an increase in average collagen fibre diameter. Minor changes in biomaterial mechanics result in significant changes in programmed cell behaviour, resulting in adoption of markedly different cell morphologies and ability to remodel the scaffold. Advanced understanding of cell–biomaterial interactions, over short and long-term culture, is of critical importance in the development of novel tissue engineering strategies for the fabrication of biomimetic 3D neuro-tissue constructs. Simple methods of tailoring the initial mechanical environment presented to SH-SY5Y populations in 3D can lead to significantly different programs of network development over time.