Actin is a versatile and ubiquitous cytoskeletal protein that plays a major role in both the establishment and the maintenance of neuronal polarity. For a long time, the most prominent roles that were attributed to actin in neurons were the movement of growth cones, polarized cargo sorting at the axon initial segment, and the dynamic plasticity of dendritic spines, since those compartments contain large accumulations of actin filaments (F-actin) that can be readily visualized using electron- and fluorescence microscopy. With the development of super-resolution microscopy in the past few years, previously unknown structures of the actin cytoskeleton have been uncovered: a periodic lattice consisting of actin and spectrin seems to pervade not only the whole axon, but also dendrites and even the necks of dendritic spines. Apart from that striking feature, patches of F-actin and deep actin filament bundles have been described along the lengths of neurites. So far, research has been focused on the specific roles of actin in the axon, while it is becoming more and more apparent that in the dendrite, actin is not only confined to dendritic spines, but serves many additional and important functions. In this review, we focus on recent developments regarding the role of actin in dendrite morphology, the regulation of actin dynamics by internal and external factors, and the role of F-actin in dendritic protein trafficking.
Compartmentalization of calcium-dependent plasticity allows for rapid actin remodeling in dendritic spines. However, molecular mechanisms for the spatio-temporal regulation of filamentous actin (F-actin) dynamics by spinous Ca-transients are still poorly defined. We show that the postsynaptic Ca sensor caldendrin orchestrates nano-domain actin dynamics that are essential for actin remodeling in the early phase of long-term potentiation (LTP). Steep elevation in spinous [Ca] disrupts an intramolecular interaction of caldendrin and allows cortactin binding. The fast on and slow off rate of this interaction keeps cortactin in an active conformation, and protects F-actin at the spine base against cofilin-induced severing. Caldendrin gene knockout results in higher synaptic actin turnover, altered nanoscale organization of spinous F-actin, defects in structural spine plasticity, LTP, and hippocampus-dependent learning. Collectively, the data indicate that caldendrin-cortactin directly couple [Ca] to preserve a minimal F-actin pool that is required for actin remodeling in the early phase of LTP.
Entry into mitosis is mediated by the phosphorylation of key cell cycle regulators by cyclin-dependent kinase 1 (Cdk1). In Xenopus embryos, the M-phase-promoting activity of Cdk1 is antagonized by protein phosphatase PP2A-B55. Hence, to ensure robust cell cycle transitions, Cdk1 and PP2A-B55 must be regulated so that their activities are mutually exclusive. The mechanism underlying PP2A-B55 inactivation at mitotic entry is well understood: Cdk1-activated Greatwall (Gwl) kinase phosphorylates Ensa/Arpp19, thereby enabling them to bind to and inhibit PP2A-B55. However, the re-activation of PP2A-B55 during mitotic exit, which is essential for cell cycle progression, is less well understood. Here, we identify protein phosphatase PP1 as an essential component of the PP2A-B55 re-activation pathway in Xenopus embryo extracts. PP1 initiates the re-activation of PP2A-B55 by dephosphorylating Gwl. We provide evidence that PP1 targets the auto-phosphorylation site of Gwl, resulting in efficient Gwl inactivation. This step is necessary to facilitate subsequent complete dephosphorylation of Gwl by PP2A-B55. Thus, by identifying PP1 as the phosphatase initiating Gwl inactivation, our study provides the molecular explanation for how Cdk1 inactivation is coupled to PP2A-B55 re-activation at mitotic exit.
Organelle positioning within neurites is required for proper neuronal function. In dendrites, with their complex cytoskeletal organization, transport of organelles is guided by local specializations of the microtubule and actin cytoskeleton, and by coordinated activity of different motor proteins. Here, we focus on the actin cytoskeleton in the dendritic shaft and describe dense structures consisting of longitudinal and branched actin filaments. These actin patches are devoid of microtubules and are frequently located at the base of spines, or form an actin mesh around excitatory shaft synapses. Using lysosomes as an example, we demonstrate that the presence of actin patches has a strong impact on dendritic organelle transport, as lysosomes frequently stall at these locations. We provide mechanistic insights on this pausing behavior, demonstrating that actin patches form a physical barrier for kinesin‐driven cargo. In addition, we identify myosin Va as an active tether which mediates long‐term stalling. This correlation between the presence of actin meshes and halting of organelles could be a generalized principle by which synapses control organelle trafficking.
The spine apparatus (SA) is an endoplasmic reticulum-related organelle that is present in a subset of dendritic spines in cortical and pyramidal neurons, and plays an important role in Ca2+ homeostasis and dendritic spine plasticity. The protein synaptopodin is essential for the formation of the SA and is widely used as a maker for this organelle. However, it is still unclear which factors contribute to its localization at selected synapses, and how it triggers local SA formation. In this study, we characterized development, localization and mobility of synaptopodin clusters in hippocampal primary neurons, as well as the molecular dynamics within these clusters. Interestingly, synaptopodin at the shaft-associated clusters is less dynamic than at spinous clusters. We identify the actin-based motor proteins myosin V (herein referring to both the myosin Va and Vb forms) and VI as novel interaction partners of synaptopodin, and demonstrate that myosin V is important for the formation and/or maintenance of the SA. We found no evidence of active microtubule-based transport of synaptopodin. Instead, new clusters emerge inside spines, which we interpret as the SA being assembled on-site.
Regulation of actin cytoskeleton dynamics in dendritic spines is crucial for learning and memory formation. Hence, defects in the actin cytoskeleton pathways are a biological trait of several brain diseases, including Alzheimer's Disease. Here, we describe a novel synaptic mechanism governed by the cyclase-associated protein 2 (CAP2), which is required for structural plasticity phenomena and completely disrupted in Alzheimer's Disease. We report that the formation of CAP2 dimers through its Cys32 is important for CAP2 binding to cofilin and for actin turnover. The Cys32-dependent CAP2 homodimerization and association to cofilin are triggered by long-term potentiation and are required for long-term potentiation-induced cofilin translocation into spines, spine remodelling and the potentiation of synaptic transmission. This mechanism is specifically affected in the hippocampus, but not in the superior frontal gyrus, of both Alzheimer's Disease patients and APP/PS1 mice, where CAP2 is down-regulated and CAP2 dimer synaptic levels are reduced. Notably, CAP2 levels in the cerebrospinal fluid are significantly increased in Alzheimer's Disease patients but not in subjects affected by frontotemporal dementia. In Alzheimer's Disease hippocampi, cofilin association to CAP2 dimer/monomer is altered and cofilin is aberrantly localized in spines. Taken together, these results provide novel insights into structural plasticity mechanisms that are defective in Alzheimer's Disease.
BackgroundRetinoid therapy is widely employed in clinical oncology to differentiate malignant cells into their more benign counterparts. However, certain high-risk cohorts, such as patients with MYCN-amplified neuroblastoma, are innately resistant to retinoid therapy. Therefore, we employed a precision medicine approach to globally profile the retinoid signalling response and to determine how an excess of cellular MYCN antagonises these signalling events to prevent differentiation and confer resistance.MethodsWe applied RNA sequencing (RNA-seq) and interaction proteomics coupled with network-based systems level analysis to identify targetable vulnerabilities of MYCN-mediated retinoid resistance. We altered MYCN expression levels in a MYCN-inducible neuroblastoma cell line to facilitate or block retinoic acid (RA)-mediated neuronal differentiation. The relevance of differentially expressed genes and transcriptional regulators for neuroblastoma outcome were then confirmed using existing patient microarray datasets.ResultsWe determined the signalling networks through which RA mediates neuroblastoma differentiation and the inhibitory perturbations to these networks upon MYCN overexpression. We revealed opposing regulation of RA and MYCN on a number of differentiation-relevant genes, including LMO4, CYP26A1, ASCL1, RET, FZD7 and DKK1. Furthermore, we revealed a broad network of transcriptional regulators involved in regulating retinoid responsiveness, such as Neurotrophin, PI3K, Wnt and MAPK, and epigenetic signalling. Of these regulators, we functionally confirmed that MYCN-driven inhibition of transforming growth factor beta (TGF-β) signalling is a vulnerable node of the MYCN network and that multiple levels of cross-talk exist between MYCN and TGF-β. Co-targeting of the retinoic acid and TGF-β pathways, through RA and kartogenin (KGN; a TGF-β signalling activating small molecule) combination treatment, induced the loss of viability of MYCN-amplified retinoid-resistant neuroblastoma cells.ConclusionsOur approach provides a powerful precision oncology tool for identifying the driving signalling networks for malignancies not primarily driven by somatic mutations, such as paediatric cancers. By applying global omics approaches to the signalling networks regulating neuroblastoma differentiation and stemness, we have determined the pathways involved in the MYCN-mediated retinoid resistance, with TGF-β signalling being a key regulator. These findings revealed a number of combination treatments likely to improve clinical response to retinoid therapy, including co-treatment with retinoids and KGN, which may prove valuable in the treatment of high-risk MYCN-amplified neuroblastoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-017-0407-3) contains supplementary material, which is available to authorized users.
High expression of the actin bundling protein Fascin increases the malignancy of tumor cells. Here we show that fascin expression is up-regulated in more malignant sub-cell lines of MDA-MB-231 cells as compared to parental cells. Since also parental MDA-MB-231 cells exhibit high fascin levels, increased fascin expression was termed as “hyperexpression”. To examine the effect of fascin hyperexpression, fascin was hyperexpressed in parental MDA-MB-231 cells and metastasis was analyzed in NOD scid gamma (NSG) mice. In addition, the effect of fascin mutants with inactive or constitutively active actin bundling activity was examined. Unexpectedly, we found that hyperexpression of both, wildtype (wt) and mutant fascin strongly increased metastasis in vivo, showing that the effect of fascin hyperexpression did not depend on its actin bundling activity. Cellular assays revealed that hyperexpression of wt and mutant fascin increased adhesion of MDA-MB-231 cells while transmigration and proliferation were not affected. Since it has been shown that fascin controls adhesion by directly interacting with microtubules (MTs), we analyzed if fascin hyperexpression affects MT dynamics. We found that at high concentrations fascin significantly increased MT dynamics in cells and in cell-free approaches. In summary our data show that strong expression of fascin in breast cancer cells increases metastasis independent of its actin bundling activity. Thus, it seems that the mechanism of fascin-stimulated metastasis depends on its concentration.
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