The mammalian endocytic cytoskeleton

Clathrin-mediated endocytosis (CME) is the major route through which cells internalise various substances and recycle membrane components. Via the coordinated action of many proteins, the membrane bends and in-vaginates to form a vesicle that buds off-along with its contents-into the cell. The contribution of the actin cytoskeleton to this highly dynamic process in mammalian cells is not well understood. Unlike in yeast, where there is a strict requirement for actin in CME, the significance of the actin cytoskeleton to mammalian CME is variable. However, a growing number of studies have established the actin cytoskeleton as a core component of mammalian CME, and our understanding of its contribution has been increasing at a rapid pace. In this review, we summarise the state-of-the-art regarding our understanding of the endocytic cytoskeleton, its physiological significance, and the questions that remain to be answered.Peer reviewe

ASD-Associated De Novo Mutations in Five Actin Regulators Show Both Shared and Distinct Defects in Dendritic Spines and Inhibitory Synapses in Cultured Hippocampal Neurons

Many actin cytoskeleton-regulating proteins control dendritic spine morphology and density, which are cellular features often altered in autism spectrum disorder (ASD). Recent studies using animal models show that autism-related behavior can be rescued by either manipulating actin regulators or by reversing dendritic spine density or morphology. Based on these studies, the actin cytoskeleton is a potential target pathway for developing new ASD treatments. Thus, it is important to understand how different ASD-associated actin regulators contribute to the regulation of dendritic spines and how ASD-associated mutations modulate this regulation. For this study, we selected five genes encoding different actin-regulating proteins and induced ASD-associated de novo missense mutations in these proteins. We assessed the functionality of the wild-type and mutated proteins by analyzing their subcellular localization, and by analyzing the dendritic spine phenotypes induced by the expression of these proteins. As the imbalance between excitation and inhibition has been suggested to have a central role in ASD, we additionally evaluated the density, size and subcellular localization of inhibitory synapses. Common for all the proteins studied was the enrichment in dendritic spines. ASD-associated mutations induced changes in the localization of alpha-actinin-4, which localized less to dendritic spines, and for SWAP-70 and SrGAP3, which localized more to dendritic spines. Among the wild-type proteins studied, only alpha-actinin-4 expression caused a significant change in dendritic spine morphology by increasing the mushroom spine density and decreasing thin spine density. We hypothesized that mutations associated with ASD shift dendritic spine morphology from mushroom to thin spines. An M554V mutation in alpha-actinin-4 (ACTN4) resulted in the expected shift in dendritic spine morphology by increasing the density of thin spines. In addition, we observed a trend toward higher thin spine density withmutations inmyosin IXb and SWAP-70. Myosin IIb and myosin IXb expression increased the proportion of inhibitory synapses in spines. The expression of mutated myosin IIb (Y265C), SrGAP3 (E469K), and SWAP-70 (L544F) induced variable changes in inhibitory synapses.Peer reviewe

Tropomyosin Tpm3.1 is required to maintain the structure and function of the axon initial segment

The axon initial segment (AIS) is the site of action potential initiation and serves as a cargo transport filter and diffusion barrier that helps maintain neuronal polarity. The AIS actin cytoskeleton comprises actin patches and periodic sub-membranous actin rings. We demonstrate that tropomyosin isoform Tpm3.1 co-localizes with actin patches and that the inhibition of Tpm3.1 led to a reduction in the density of actin patches. Furthermore, Tpm3.1 showed a periodic distribution similar to sub-membranous actin rings but Tpm3.1 was only partially congruent with sub-membranous actin rings. Nevertheless, the inhibition of Tpm3.1 affected the uniformity of the periodicity of actin rings. Furthermore, Tpm3.1 inhibition led to reduced accumulation of AIS structural and functional proteins, disruption in sorting somatodendritic and axonal proteins, and a reduction in firing frequency. These results show that Tpm3.1 is necessary for the structural and functional maintenance of the AIS.Peer reviewe

The Structure and Dynamics of the Actin Cytoskeleton in the Axon Initial Segment

The axon initial segment (AIS) is the site of action potential initiation and plays an important role in maintaining neuronal polarity. Recent advances in super-resolution microscopy revealed the presence of an intricate membrane-associated periodic lattice in the AIS that contains sub-membranous actin rings periodically spaced ~190 nm and connected to a spectrin-ankyrin lattice. The precise function of these actin rings in unclear, as well as details of their structure and dynamics. The insensitivity of the AIS to actin-disrupting drugs led to the long-held view that actin is not a critical component of AIS structure. Here I show that the AIS contains a population of relatively stable, latrunculin-resistant actin filaments that are decorated by the tropomyosin isoform Tpm3.1. Disrupting these filaments through the perturbation of Tpm3.1 function led to the loss of accumulation of ankyrin G and other AIS markers, disruption of neuronal polarity, the loss of the clustering of voltage-gated sodium channels, and a rapid reduction in firing frequency. The findings I present in this thesis suggest that actin plays an important role in maintaining AIS structure and function, more important than previously appreciated.Hermosolut ovat pitkälle erikoistuneita soluja ja niiden eri haarakkeilla on erilaiset tehtävät. Tuojahaarakkeet tuovat signaalin muista hermosoluista hermosolun keskukseen, soomaan. Soomasta lähtee viejähaarake, joka vie signaalin taas eteenpäin. Viejähaarakkeen alussa, heti soomasta lähtiessä, on erityinen alue, jota suomeksi kutsutaan viejähaarakkeen alkuosaksi (englanniksi axon initial segment). Viejähaarakkeen alkuosa on tärkeä, sillä siinä päätetään, lähteekö signaali eteenpäin seuraaville hermosoluille. Viejähaarakkeen alkuosa myös säätelee mitkä solun rakennusaineet kuljetetaan viejähaarakkeisiin ja mitkä pidetään soomassa tai tuojahaarakkeissa. Näin solu pystyy erilaistamaan solun eri osat eri tehtäviä varten, tuoja- ja viejähaarakkeet on rakennettu eri rakennuskomponenteista. Viejähaarakkeen rakenne ja proteiinien järjestys on ensiarvoisen tärkeää viejähaarakkeen alkuosan tehokkaalle toiminnalle. Mikroskopiatekniikoiden kehittyminen viime vuosina on mahdollistanut viejähaarakkeen aktiinitukirangan rakenteen tarkemman tarkastelun. Aktiinitukiranka on solujen luusto ja lihakset, aktiinimolekyylit järjestäytyvät aktiinisäikeiksi, joista voi rakentaa soluun erilaisia rakenteita. Säätelystä riippuu miten pysyviä tai dynaamisia nämä rakenteet ovat. Väitöskirjassani selvitin viejähaarakkeen aktiinitukirangan rakennetta ja aktiinisäikeiden dynamiikkaa. Havaitsin että viejähaarakkeen alkuosan aktiinisäikeet ovat hyvin stabiileja. Tutkin myös miten aktiinisäikeisiin sitoutuva tropomyosiini 3.1 proteiini säätelee viejähaarakkeen alkuosan aktiinitukirankaa. Yllätyksekseni tämä tropomyosiini oli hyvin keskeinen koko viejähaarakkeen alkuosan rakenteen ylläpidolle. Kun poistin tropomyosiini 3.1 proteiinin, tai estin sen toimintaa hermosoluissa, tuhosin samalla viejähaarakkeen rakenteen ja toimivuuden, sekä rakennuskomponenttien kuljetuksen säätelyn, että signaalin eteenpäin viemisen. Aiemmin luultiin että aktiinitukiranka ei ole tärkeä viejähaarakkeen alkuosan rakenteelle tai toiminnalle. Tulokseni kuitenkin osoittavat, että aktiinitukirangan normaali rakenne on edellytys koko viejähaarakkeen rakenteen ja toiminnan ylläpidolle

Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy (3D-SIM)

Funding Information: Dr. Pirta Hotulainen is acknowledged for her critical comments, invaluable for preparing this manuscript. Dr. Rimante Minkeviciene is acknowledged for her help in preparing the neuronal cultures used for the original experiments. All imaging was performed in the Biomedicum Imaging Unit. This work was supported by the Academy of Finland (D.M., SA 266351) and Doctoral Programme Brain & Mind (A.A.) Funding Information: Dr. Pirta Hotulainen is acknowledged for her critical comments, invaluable for preparing this manuscript. Dr. Rimante Minkeviciene is acknowledged for her help in preparing the neuronal cultures used for the original experiments. All imaging was performed in the Biomedicum Imaging Unit. This work was supported by the Academy of Finland (D.M., SA 266351) and Doctoral Programme Brain & Mind (A.A.). Publisher Copyright: © 2022, Journal of Visualized Experiments. All rights reserved.The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to the maintenance of neuronal polarity by sorting somato-dendritic cargo. A membrane periodic skeleton (MPS) comprising periodic actin rings provides a scaffold for anchoring various AIS proteins, including structural proteins and different ion channels. Although recent proteomic approaches have identified a considerable number of novel AIS components, details of the structure of the MPS and the roles of its individual components are lacking. The distance between individual actin rings in the MPS (~190 nm) necessitates the employment of super-resolution microscopy techniques to resolve the structural details of the MPS. This protocol describes a method for using cultured rat hippocampal neurons to examine the precise localization of an AIS protein in the MPS relative to sub-membranous actin rings using 3D-structured illumination microscopy (3D-SIM). In addition, an analytical approach to quantitively assess the periodicity of individual components and their position relative to actin rings is also described.Peer reviewe

Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin alpha 5 beta 1

Hakanpaa et al. show that reticular adhesions are assembled at flat clathrin lattices, a process inhibited by fibronectin and its receptor, integrin alpha 5 beta 1. This novel adhesion assembly mechanism is coupled to cell migration and reveals a unique crosstalk between cell-matrix adhesions.Reticular adhesions (RAs) consist of integrin alpha v beta 5 and harbor flat clathrin lattices (FCLs), long-lasting structures with similar molecular composition as clathrin-mediated endocytosis (CME) carriers. Why FCLs and RAs colocalize is not known. Here, we show that RAs are assembled at FCLs in a process controlled by fibronectin (FN) and its receptor, integrin alpha 5 beta 1. We observed that cells on FN-rich matrices displayed fewer FCLs and RAs. CME machinery inhibition abolished RAs and live-cell imaging showed that RA establishment requires FCL coassembly. The inhibitory activity of FN was mediated by the activation of integrin alpha 5 beta 1 at Tensin1-positive fibrillar adhesions. Conventionally, endocytosis disassembles cellular adhesions by internalizing their components. Our results present a novel paradigm in the relationship between these two processes by showing that endocytic proteins can actively function in the assembly of cell adhesions. Furthermore, we show this novel adhesion assembly mechanism is coupled to cell migration via unique crosstalk between cell-matrix adhesions.Peer reviewe

Tropomyosin Tpm3.1 Is Required to Maintain the Structure and Function of the Axon Initial Segment

The axon initial segment (AIS) is the site of action potential initiation and serves as a cargo transport filter and diffusion barrier that helps maintain neuronal polarity. The AIS actin cytoskeleton comprises actin patches and periodic sub-membranous actin rings. We demonstrate that tropomyosin isoform Tpm3.1 co-localizes with actin patches and that the inhibition of Tpm3.1 led to a reduction in the density of actin patches. Furthermore, Tpm3.1 showed a periodic distribution similar to sub-membranous actin rings but Tpm3.1 was only partially congruent with sub-membranous actin rings. Nevertheless, the inhibition of Tpm3.1 affected the uniformity of the periodicity of actin rings. Furthermore, Tpm3.1 inhibition led to reduced accumulation of AIS structural and functional proteins, disruption in sorting somatodendritic and axonal proteins, and a reduction in firing frequency. These results show that Tpm3.1 is necessary for the structural and functional maintenance of the AIS