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The balance of actin filament polymerization and depolymerization maintains a steady state network treadmill in neuronal growth cones essential for motility and guidance. Here we have investigated the connection between depolymerization and treadmilling dynamics. We show that polymerization-competent barbed ends are concentrated at the leading edge and depolymerization is distributed throughout the peripheral domain. We found a high-to-low G-actin gradient between peripheral and central domains. Inhibiting turnover with jasplakinolide collapsed this gradient and lowered leading edge barbed end density. Ultrastructural analysis showed dramatic reduction of leading edge actin filament density and filament accumulation in central regions. Live cell imaging revealed that the leading edge retracted even as retrograde actin flow rate decreased exponentially. Inhibition of myosin II activity before jasplakinolide treatment lowered baseline retrograde flow rates and prevented leading edge retraction. Myosin II activity preferentially affected filopodial bundle disassembly distinct from the global effects of jasplakinolide on network turnover. We propose that growth cone retraction following turnover inhibition resulted from the persistence of myosin II contractility even as leading edge assembly rates decreased. The buildup of actin filaments in central regions combined with monomer depletion and reduced polymerization from barbed ends suggests a mechanism for the observed exponential decay in actin retrograde flow. Our results show that growth cone motility is critically dependent on continuous disassembly of the peripheral actin network.

Interactions between microtubules and actin are a basic phenomenon that underlies many fundamental processes in which dynamic cellular asymmetries need to be established and maintained. These are processes as diverse as cell motility, neuronal pathfinding, cellular wound healing, cell division and cortical flow. Microtubules and actin exhibit two mechanistic classes of interactions — regulatory and structural. These interactions comprise at least three conserved 'mechanochemical activity modules' that perform similar roles in these diverse cell functions.

Adhesions are multi-molecular complexes that transmit forces generated by a cell's acto-myosin networks to external substrates. While the physical properties of some of the individual components of adhesions have been carefully characterized, the mechanics of the coupling between the cytoskeleton and the adhesion site as a whole are just beginning to be revealed. We characterized the mechanics of nascent adhesions mediated by the immunoglobulin-family cell adhesion molecule apCAM, which is known to interact with actin filaments. Using simultaneous visualization of actin flow and quantification of forces transmitted to apCAM-coated beads restrained with an optical trap, we found that adhesions are dynamic structures capable of transmitting a wide range of forces. For forces in the picoNewton scale, the nascent adhesions' mechanical properties are dominated by an elastic structure which can be reversibly deformed by up to 1 µm. Large reversible deformations rule out an interface between substrate and cytoskeleton that is dominated by a number of stiff molecular springs in parallel, and favor a compliant cross-linked network. Such a compliant structure may increase the lifetime of a nascent adhesion, facilitating signaling and reinforcement.

Filopodial actin bundles guide microtubule assembly in the growth cone peripheral (P) domain and retrograde actin-network flow simultaneously transports microtubules rearward. Therefore, microtubule-end position is determined by the sum of microtubule assembly and retrograde transport rates. However, how filopodia actually affect microtubule assembly dynamics is unknown. To address this issue we quantitatively assessed microtubule and actin dynamics before and after selective removal of filopodia. Filopodium removal had surprisingly little effect on retrograde actin-flow rates or underlying network structures, but resulted in an approximate doubling of peripheral microtubule density and deeper penetration of microtubules into the P domain. The latter stemmed from less efficient coupling of microtubules to remaining actin networks and not from a change in microtubule polymer dynamics. Loss of filopodia also resulted in increased lateral microtubule movements and a more randomized microtubule distribution in the P domain. In summary, filopodia do not seem to be formally required for microtubule advance; however, their presence ensures radial distribution of microtubules in the P domain and facilitates microtubule transport by retrograde flow. The resulting dynamic steady state has interesting implications for rapid microtubule-positioning responses in the P domain.

Invasively migrating cells thread their nucleus through confined interstitial spaces. How cells protect the nucleus from intracellular forces is poorly understood. Here, we show that the septin cytoskeleton buffers the actomyosin forces that power nuclear movement. Septin filaments comprising SEPT9, a septin amplified in breast cancer, align with perinuclear actomyosin cables which exhibit higher tensile stress during 3D confined migration through narrower pores. SEPT9 depletion amplifies actin stress during confined migration and after myosin II hyper-activation in non-migrating cells causing actin and nuclear envelope ruptures. Following confined migration, DNA breaks, nuclear blebs, micronuclei and cell death increase in SEPT9-depleted cells, phenotypes rescued by the oncogenic SEPT9 isoform 1. Clinicogenomic data reveal that amplification associates with lower genomic alteration in aggressive breast tumors and higher patient mortality. We propose that septins are a mechanoprotective element of the cytoskeleton, and SEPT9 amplification enhances tumor cell survival by preventing nuclear damage.

Septins are cytoskeletal filaments that associate with the actin and microtubule cytoskeleton, but the mechanisms that govern septin crosstalk and function with these networks are largely unknown. Here, we show that glycogen synthase kinase 3 (GSK3) directly phosphorylates septin-9 (SEPT9), acting as a molecular switch that bidirectionally controls septin distribution between actin and microtubules. We show that GSK3 inhibition redistributes endogenous SEPT9 toward microtubules in multiple cell types. Phosphomimetic mutations at serines 82 and 85 reduce microtubule binding and enhance actin association in cells and in vitro, while phosphonull mutations promote microtubule binding and growth. In primary hippocampal neurons, GSK3β inactivation promotes SEPT9-microtubule association, and phosphomimetic mutations impair asymmetric neurite growth during neuronal polarization. These findings reveal a phosphorylation-dependent mechanism of septin partitioning between actin and microtubules, placing the cytoskeletal functions of septins under the control of GSK3 - a kinase linked to multiple signaling pathways of cell physiology and metabolism.

World governments have committed to halting human-induced extinctions and safeguarding important sites for biodiversity by 2020, but the financial costs of meeting these targets are largely unknown. We estimate the cost of reducing the extinction risk of all globally threatened bird species (by > 1 International Union for Conservation of Nature Red List category) to be U.S. $0.875 to $1.23 billion annually over the next decade, of which 12% is currently funded. Incorporating threatened nonavian species increases this total to U.S. $3.41 to $4.76 billion annually. We estimate that protecting and effectively managing all terrestrial sites of global avian conservation significance (11,731 Important Bird Areas) would cost U.S. $ 65.1 billion annually. Adding sites for other taxa increases this to U.S. $76.1 billion annually. Meeting these targets will require conservation funding to increase by at least an order of magnitude.

Mitochondrial functions are intrinsically linked to their morphology and membrane ultrastructure. Characterizing abnormal mitochondrial structural features may thus provide insight into the underlying pathogenesis of inherited and acquired mitochondrial diseases. Following a systematic literature review on ultrastructural defects in mitochondrial myopathy, we investigated skeletal muscle biopsies from seven subjects with genetically defined mtDNA mutations. Mitochondrial ultrastructure and morphology were characterized using two complimentary approaches: transmission electron microscopy (TEM) and serial block face scanning EM (SBF-SEM) with 3D reconstruction. Six ultrastructural abnormalities were identified including i) paracrystalline inclusions, ii) linearization of cristae and abnormal angular features, iii) concentric layering of cristae membranes, iv) matrix compartmentalization, v) nanotunelling and vi) donut-shaped mitochondria. In light of recent molecular advances in mitochondrial biology, these findings reveal novel aspects of mitochondrial ultrastructure and morphology in human tissues with implications for understanding the mechanisms linking mitochondrial dysfunction to disease.

Nucleic acid-based vaccines such as viral vectors, plasmid DNA, and mRNA are being developed as a means to address a number of unmet medical needs that current vaccine technologies have been unable to address. Here, we describe a cationic nanoemulsion (CNE) delivery system developed to deliver a self-amplifying mRNA vaccine. This nonviral delivery system is based on Novartis's proprietary adjuvant MF59, which has an established clinical safety profile and is well tolerated in children, adults, and the elderly. We show that nonviral delivery of a 9 kb self-amplifying mRNA elicits potent immune responses in mice, rats, rabbits, and nonhuman primates comparable to a viral delivery technology, and demonstrate that, relatively low doses (75 µg) induce antibody and T-cell responses in primates. We also show the CNE-delivered self-amplifying mRNA enhances the local immune environment through recruitment of immune cells similar to an MF59 adjuvanted subunit vaccine. Lastly, we show that the site of protein expression within the muscle and magnitude of protein expression is similar to a viral vector. Given the demonstration that self-amplifying mRNA delivered using a CNE is well tolerated and immunogenic in a variety of animal models, we are optimistic about the prospects for this technology.

Mitochondrial disease associated with the pathogenic m.3243A>G variant is a common, clinically heterogeneous, neurogenetic disorder. Using multiple linear regression and linear mixed modelling, we evaluated which commonly assayed tissue (blood N  = 231, urine N  = 235, skeletal muscle N  = 77) represents the m.3243A>G mutation load and mitochondrial DNA (mtDNA) copy number most strongly associated with disease burden and progression. m.3243A>G levels are correlated in blood, muscle and urine ( R 2  = 0.61–0.73). Blood heteroplasmy declines by ~2.3%/year; we have extended previously published methodology to adjust for age. In urine, males have higher mtDNA copy number and ~20% higher m.3243A>G mutation load; we present formulas to adjust for this. Blood is the most highly correlated mutation measure for disease burden and progression in m.3243A>G‐harbouring individuals; increasing age and heteroplasmy contribute ( R 2  = 0.27, P  < 0.001). In muscle, heteroplasmy, age and mtDNA copy number explain a higher proportion of variability in disease burden ( R 2  = 0.40, P  < 0.001), although activity level and disease severity are likely to affect copy number. Whilst our data indicate that age‐corrected blood m.3243A>G heteroplasmy is the most convenient and reliable measure for routine clinical assessment, additional factors such as mtDNA copy number may also influence disease severity. Synopsis The m.3243A>G pathogenic mtDNA variant is associated with a highly heterogeneous multisystem disorder and varying mutation levels across tissues. In this study, mutation levels were characterised in three commonly sampled tissues ‐ blood, urine, skeletal muscle ‐ and correlated with disease burden. Urine m.3243A>G heteroplasmy levels display more variability than blood levels and must be corrected for a ˜20% lower level in females. Blood m.3243A>G heteroplasmy levels must be corrected for a decline of ˜2.3% per year. Disease burden and progression are more strongly associated with blood m.3243A>G heteroplasmy levels than urine levels. 27% of the variance in disease burden can be attributed to blood m.3243A>G heteroplasmy and age. Age, m.3243A>G heteroplasmy level and mtDNA copy number in skeletal muscle explain 40% of the variance in disease burden. Graphical Abstract The m.3243A>G pathogenic mtDNA variant is associated with a highly heterogeneous multisystem disorder and varying mutation levels across tissues. In this study, mutation levels were characterised in three commonly sampled tissues ‐ blood, urine, skeletal muscle ‐ and correlated with disease burden.