The actin cytoskeleton plays essential roles in cell polarization and cell morphogenesis of the budding yeast Saccharomyces cerevisiae. Yeast cells utilize formin-generated actin cables as tracks for polarized transport, which forms the basis for a positive feedback loop driving Cdc42-dependent cell polarization. Previous studies on cable organization mostly focused on polarized actin cables in budded cells and their role as relatively static tracks for myosin-dependent organelle transport. Using quantitative live cell imaging, we have recently characterized the dynamics of cortical actin cables throughout the yeast cell cycle. Surprisingly, randomly oriented actin cables in G1 cells exhibited the highest level of dynamics, while cable dynamics was markedly slowed down upon cell polarization. We further demonstrated that the rapid dynamics of randomly oriented cables were driven by the formin Bni1 and Myosin V. Our data suggested a precise spatio-temporal regulation of the two yeast formins, as well as an unexpected mechanism of actin cable rearrangement through myosins. Here we discuss the immediate significance of these findings, which illustrates the importance of generating randomness for cellular organization.
actin; formin; myosin; polarity; self organization
Adherens junctions (AJs) and tight junctions (TJs) represent key adhesive structures that regulate the apico-basal polarity and barrier properties of epithelial layers. AJs and TJs readily undergo disassembly and reassembly during normal tissue remodeling and disruption of epithelial barriers in diseases. Such junctional plasticity depends on the orchestrated dynamics of the plasma membrane with its underlying F-actin cytoskeleton, however the interplay between these cellular structures remains poorly understood. Recent studies highlighted the spectrin-adducin-based membrane skeleton as an emerging regulator of AJ and TJ integrity and remodeling. Here we discuss new evidences implicating adducin, spectrin and other membrane skeleton proteins in stabilization of epithelial junctions and regulation of junctional dynamics. Based on the known ability of the membrane skeleton to link cortical actin filaments to the plasma membrane, we hypothesize that the spectrin-adducin network serves as a critical signal and force transducer from the actomyosin cytoskeleton to junctions during remodeling of AJs and TJs.
adherens junctions; tight junctions; permeability; membrane skeleton; actomyosin; contractility; calcium switch
The function of nuclear actin is poorly understood. It is known to be a discrete component of several chromatin-modifying complexes. Nevertheless, filamentous forms of actin are important for various nuclear processes as well. Nuclear actin is often associated with nuclear actin-related protein Arp4 and other actin-related proteins like Arp8 in the INO80 chromatin remodeler. We recently determined the crystal structure of S. cerevisiae Arp4 that explains why Arp4 is unable to form actin like filaments and shows that it is constitutively bound to an ATP nucleotide. More interestingly, in vitro activities of Arp4 and Arp8 seem to be directed towards stabilizing monomeric actin and to integrate it stoichiometrically into the INO80 complex. Based on this activity, we discuss possible roles of nuclear Arps in chromatin modifying complexes and in regulating more general aspects of nuclear actin dynamics.
actin-related proteins; chromatin remodeling; INO80 complex; nuclear actin
Cilia are protrusions on the surface of cells. They are frequently motile and function to propel cells in an aqueous environment or to generate fluid flow. Equally important is the role of immotile cilia in detecting environmental changes or in sensing extracellular signals. The structure of cilia is supported by microtubules, and their formation requires microtubule-dependent motors, kinesins, which are thought to transport both structural and signaling ciliary proteins from the cell body into the distal portion of the ciliary shaft. In multicellular organisms, multiple kinesins are known to drive ciliary transport, and frequently cilia of a single cell type require more than one kinesin for their formation and function. In addition to kinesin-2 family motors, which function in cilia of all species investigated so far, kinesins from other families contribute to the transport of signaling proteins in a tissue-specific manner. It is becoming increasingly obvious that functional relationships between ciliary kinesins are complex, and a good understanding of these relationships is essential to comprehend the basis of biological processes as diverse as olfaction, vision, and embryonic development.
C. elegans; cilia; flagella; intraflagellar; kinesin; motor; mouse; photoreceptor; zebrafish
The PCI fold is based on a stack of α-helices topped with a winged-helix domain and is found in a range of proteins that form central parts of large complexes such as the proteasome lid, the COP9 signalosome, elongation factor eIF3, and the TREX-2 complex. Recent structural determinations have given intriguing insight into how these folds function both to facilitate the generation of larger proteinaceous assembles and also to interact functionally with nucleic acids.
COP9 complex; PCI fold; PCI protein; TREX-2 complex; eIF3; proteasome
The unique innovation of the layered neocortex in mammalian evolution is believed to facilitate adaptive radiation of mammalian species to various ecological environments by furnishing high information processing ability. There are no transitional states from the non-mammalian simple brain to the mammalian multilayered neocortex, and thus it is totally a mystery so far how this brain structure has been acquired during evolution. In our recent study, we found the evidence showing that the evolutionary origin of the neocortical neuron subtypes predates the actual emergence of layer structure. Our comparative developmental analysis of the chick pallium, homologous to the mammalian neocortex, revealed that mammals and avians fundamentally share the neocortical neuron subtypes and their production mechanisms, suggesting that their common ancestor already possessed a similar neuronal repertory. We further demonstrated that the neocortical layer-specific neuron subtypes are arranged as mediolaterally separated domains in the chick, but not as layers in the mammalian neocortex. These animal group-specific neuronal arrangements are accomplished by spatial modulation of the neurogenetic program, suggesting an evolutionary hypothesis that the regulatory changes in the neurogenetic program innovated the mammalian specific layered neocortex.
bird; brain patterning; evolution; layer; mammal; neocortex; neural progenitor; neuron subtype; pallium; stem cell
During mitosis, microtubules (MTs) are massively rearranged into three sets of highly dynamic MTs that are nucleated from the centrosomes to form the mitotic spindle. Tight regulation of spindle positioning in the dividing cell and chromosome alignment at the center of the metaphase spindle are required to ensure perfect chromosome segregation and to position the cytokinetic furrow that will specify the two daughter cells. Spindle positioning requires regulation of MT dynamics, involving depolymerase activities together with cortical and kinetochore-mediated pushing and pulling forces acting on astral MTs and kinetochore fibres. These forces rely on MT motor activities. Cortical pulling forces exerted on astral MTs depend upon dynein/dynactin complexes and are essential in both symmetric and asymmetric cell division. A well-established spindle positioning pathway regulating the cortical targeting of dynein/dynactin involves the conserved LGN (Leu-Gly-Asn repeat-enriched-protein) and NuMA (microtubule binding nuclear mitotic apparatus protein) complex.1 Spindle orientation is also regulated by integrin-mediated cell adhesion2 and actin retraction fibres that respond to mechanical stress and are influenced by the microenvironment of the dividing cell.3 Altering the capture of astral MTs or modulating pulling forces affects spindle position, which can impair cell division, differentiation and embryogenesis.
In this general scheme, the activity of mitotic kinases such as Auroras and Plk1 (Polo-like kinase 1) is crucial.4 Recently, the p21-activated kinases (PAKs) emerged as novel important players in mitotic progression. In our recent article, we demonstrated that PAK4 regulates spindle positioning in symmetric cell division.5 In this commentary, and in light of recent published studies, we discuss how PAK4 could participate in the regulation of mechanisms involved in spindle positioning and orientation.
astral microtubules; dynein; p21-activated kinase; spindle orientation and positioning
The THO complex is a nuclear structure whose architecture is conserved among all kingdoms and plays an important role in mRNP biogenesis connecting transcription elongation with mRNA maturation and export. Recent data indicates that the THO complex is necessary for the proper expression of some genes, assurance of genetic stability by preventing transcription-associated recombination. Yeast THO has been described as a heterotetramer (Tho2, Hpr1, Mft1 and Thp2) that performs several functions through the interaction with other proteins like Tex1 or the mRNA export factors Sub2 and Yra1, with which it forms the TRanscription and EXport complex (TREX). In this article we review the cellular role of THO, which we show to be composed of five subunits with Tex1 being also an integral part of the complex. We also show a low-resolution structure of THO and localize some of its components. We discuss the consequences of THO interaction with nucleic acids through the unfolded C-terminal region of Tho2, highlighting the importance of unfolded regions in eukaryotic proteins. Finally, we comment on THO recruitment to active chromatin, a role that is linked to mRNA biogenesis.
THO complex; TREX complex; electron microscopy; mRNA export; mRNP quality control
IQGAP1 is an important cytoskeletal regulator, known to act at the plasma membrane to bundle and cap actin filaments, and to tether the cortical actin meshwork to microtubules via plus-end binding proteins. Here we describe the novel subcellular localization of IQGAP1 at the cytoplasmic face of the nuclear envelope, where it co-located with F-actin. The IQGAP1 and F-actin staining overlapped that of microtubules at the nuclear envelope, revealing a pattern strikingly similar to that observed at the plasma membrane. In detergent-extracted cells IQGAP1 was retained at cytoskeletal structures at the nuclear envelope. This finding has new implications for involvement of IQGAP1 in cell polarization and migration events and potentially in cell cycle-associated nuclear envelope assembly/disassembly.
Cdc42; IQGAP1; Rac1; actin; cell polarization; nuclear envelope
Chromatin remodeling by the SWI/SNF complex is required to activate the transcription of myogenic-specific genes. Our work addressed the details of how SWI/SNF is recruited to myogenic regulatory regions in response to differentiation signals. Surprisingly, the muscle determination factor MyoD and the SWI/SNF subunit BAF60c form a complex on the regulatory elements of MyoD-targeted genes in myogenic precursor cells. This Brg1-devoid MyoD-BAF60c complex flags the chromatin of myogenic-differentiation genes before transcription is activated. On differentiation, BAF60c phosphorylation on a conserved threonine by p38 α kinase promotes the incorporation of MyoD-BAF60c into a Brg1-based SWI/SNF complex, which remodels the chromatin and activates transcription of MyoD-target genes. Downregulation of BAF60c expression prevents MyoD access to the chromatin and the proper loading of an active myogenic transcriptosome preventing the expression of hundreds of myogenic genes. Our data support an unprecedented two-step model by which (1) pre-assembled BAF60c-MyoD complex poises the chromatin of myogenic genes for rapid transcription; (2) chromatin-bound BAF60c “senses” the myogenic differentiation cues and recruits an active SWI/SNF complex to remodel the chromatin allowing transcriptional activation.
MyoD; BAF60c; SWI/SNF; chromatin; remodeling; transcription; myogenesis; differentiation
Apoptosis is an important biological process required for the removal of unwanted or damaged cells. Mounting evidence implicates the actin cytoskeleton as both a sensor and mediator of apoptosis. Studies also suggest that actin binding proteins (ABPs) significantly contribute to apoptosis and that actin dynamics play a key role in regulating apoptosis signaling. Changes in the organization of the actin cytoskeleton has been attributed to the process of malignant transformation and it is hypothesized that remodeling of the actin cytoskeleton may enable tumor cells to evade normal apoptotic signaling. This review aims to illuminate the role of the actin cytoskeleton in apoptosis by systematically analyzing how actin and ABPs regulate different apoptosis pathways and to also highlight the potential for developing novel compounds that target tumor-specific actin filaments.
actin; apoptosis; actin binding proteins; mitochondria; Bcl-2; cancer; multi-drug resistance
Adult skeletal muscles adapt their fiber size to workload. We show that serum response factor (Srf) is required for satellite cell-mediated hypertrophic muscle growth. Deletion of Srf from myofibers, and not satellite cells, blunts overload-induced hypertrophy, and impairs satellite cell proliferation and recruitment to pre-existing fibers. We reveal a gene network in which Srf within myofibers modulates interleukin-6 and cyclooxygenase-2/interleukin-4 expressions and therefore exerts a paracrine control of satellite cell functions. In Srf-deleted muscles, in vivo overexpression of interleukin-6 is sufficient to restore satellite cell proliferation, but not satellite cell fusion and overall growth. In contrast, cyclooxygenase-2/interleukin-4 overexpression rescues satellite cell recruitment and muscle growth without affecting satellite cell proliferation, identifying altered fusion as the limiting cellular event. These findings unravel a role for Srf in the translation of mechanical cues applied to myofibers into paracrine signals, which in turn will modulate satellite cell functions and support muscle growth.
skeletal muscle; hypertrophy; satellite cells; paracrine; transcription factor
Integrin-linked kinase (ILK), PINCH and Parvin proteins form the IPP-complex that has been established as a core component of the integrin-actin link. Our recent genetic studies on Drosophila parvin, reveal that loss of function mutant defects phenocopy those observed upon loss of ILK or PINCH in the muscle and the wing, strengthening the notion that these proteins function together in the organism. Our work identified that ILK is necessary and sufficient for parvin subcellular localization, corroborating previous data indicating a direct association between these two proteins. Further genetic epistasis analysis of the IPP-complex assembly at integrin adhesion sites reveals that depending on the cell context each component is required differently. At the muscle attachment sites of the embryo, ILK is placed upstream in the hierarchy of genetic interactions required for the IPP-complex assembly. By contrast, in the wing epithelium the three proteins are mutually interdependent. Finally, we uncovered a novel property for the CH1-domain of parvin: its recruitment at the integrin-containing junctions in an ILK-dependent manner. Apparently, this ability of the CH1-domain is controlled by the inter-CH linker region. Thus, an intramolecular interaction within parvin could serve as a putative regulatory mechanism controlling the ILK-Parvin interaction.
integrin; cell adhesion; PINCH; actin; Drosophila
Tetraspanins regulate the signaling, trafficking and biosynthetic processing of associated proteins, and may link the extracellular domain of α-chain integrins with intracellular signaling molecules, including PI4K and PKC, both of which regulate cytoskeletal architecture. We showed that TSPAN7, a member of tetraspannin-family, promotes filopodia and dendritic spine formation in cultured hippocampal neurons, and is required for spine stability and normal synaptic transmission. TSPAN7 directly interacts with the PDZ domain of protein interacting with C kinase 1 (PICK1), and associates with AMPAR subunit GluA2 and β1-integrin. TSPAN7 regulates PICK1 and GluA2/3 association, and AMPA receptor trafficking. These findings identify TSPAN7 as a key player in the morphological and functional maturation of glutamatergic synapses.
intellectual disability; AMPAR trafficking; synapse function/plasticity; tetrasapanins; TSPAN7; integrins; PICK1
Our recent paper examined how pelvic fins and their musculature form developmentally and how these mechanisms have evolved within the vertebrate lineage, a process fundamental to the tetrapod transition. The transition from the water onto the land is among one of the most well studied steps in the evolutionary history of vertebrates, yet the genetic basis of this evolutionary transition is little studied and ill-defined. The advent of these terrestrial species resulted in a shift in locomotor strategies from the rhythmic undulating muscles of the fish body to a reliance upon powerful weight bearing muscles of the limbs to generate movement. We demonstrated that the pelvic fin muscles of bony fish are generated by a mechanism that has features of both of limb/fin muscle formation in tetrapods and primitive cartilaginous fish. We hypothesize that the adoption of the fully derived mode of hindlimb muscle formation, was a further modification of the mode of development deployed to generate pelvic fin muscles, a shift in overall muscle bioarchitecture we believe was critical to the success of the tetrapod transition.
muscle; evolution; fin; limb; zebrafish; tetrapod
Scaffolding proteins that are associated with glutamate receptors in dendritic spines govern the location and function of receptors to control synaptic transmission. Unraveling the spatio-temporal dynamics of protein-protein interactions within components of the scaffolding complex will bring to light the function of these interactions. Combining bioluminescence resonance energy transfer (BRET) imaging to electrophysiological recordings, we have recently shown that GKAP, a core protein of the scaffolding complex, interacts with DLC2, a protein associated with molecular motors. Synaptic activity-induced GKAP-DLC2 interaction in spines stabilizes the scaffolding complex and enhances the NMDA currents. Interestingly, this work placed emphasis on the bioarchitectural dependence of protein-protein interaction dynamics. Depending on physiological conditions, the modulation in space and time of protein-protein interaction is acutely regulated, engendering a subtle control of synaptic transmission in the state of the individual synapse.
bioluminescence resonance energy transfer (BRET); dendritic spine; dynein light chain 2 (DLC2); glutamate receptors; guanylate kinase-associated protein (GKAP); protein-protein interaction; scaffolding proteins; synaptic transmission
Checkpoint controls, the surveillance pathways that impose “an order of execution” on the major cell cycle events, are critical to the maintenance of genome stability. When cells fail to execute a cellular event or do so erroneously due to misregulation or exposure to genotoxic stresses, these evolutionarily conserved regulatory circuits prevent passage to the subsequent event, thus bringing the cell cycle to a halt. Once the checkpoint stimulus is removed, cells recover from the arrest and eventually resume cell cycle progression. While the activation, execution and maintenance, the three major aspects of the checkpoint controls, have been investigated in detail, the recovery process remains underexplored. It is not clear if cells recover passively upon dissipation of the checkpoint signals or require an active participation by specific effectors. A recent study in the yeast Saccharomyces cerevisiae uncovered two previously unsuspected functions of Cdk1 in efficient recovery from the spindle assembly checkpoint (SAC) imposed arrest. An inability to fulfil these requirements in the absence of Cdk1 makes it virtually impossible for cells to recover from the mitotic arrest. Given the conserved nature of the SAC, these findings may have implications for vertebrate cells.
Cdk1; cell cycle; cell division; checkpoint; mitosis; recovery; spindle; yeast
Manipulation of the actin cytoskeleton is a commonly used process by which bacterial pathogens and viruses are able to neutralize host defense mechanisms and subvert them in order to replicate in a hostile environment. Diverse bacteria display a wide array of mechanisms of regulation of microfilaments to enter, move within or exit the host cell. A less studied subject is how pathogens may co-opt the actin cytoskeleton to disturb vesicle trafficking pathways, namely phagolysosomal fusion, and avoid degradation. In fact, although actin plays a role in endosomal trafficking and phagosome maturation, the knowledge on the exact mechanisms and additional players is still scarce. Recently, we found that the Legionella pneumophila virulence factor VipA is an actin nucleator, associates with actin filaments and early endosomes during infection, and interferes in yeast organelle trafficking pathways, suggesting it may be linking actin dynamics to endosome biogenesis. Further studies on this protein, together with work on other bacterial effectors, may help shed light in the role of actin in endosomal maturation.
Legionella pneumophila; Type IV Secretion System; VipA; actin; effector; multivesicular body; organelle trafficking
Regulation of the actin cytoskeleton is crucial for cell morphology and migration. One of the key molecules that regulates actin remodeling is the small GTPase Rho. Rho shuttles between the inactive GDP-bound form and the active GTP-bound form, and works as a molecular switch in actin remodeling in response to both extra- and intra-cellular stimuli. Mammalian homolog of Diaphanous (mDia) is one of the Rho effectors and produces unbranched actin filaments. While Rho GTPases activate mDia, the mechanisms of how the activity of mDia is downregulated in cells remains largely unknown. In our recent paper, we identified Liprin-α as an mDia interacting protein and found that Liprin-α negatively regulates the activity of mDia in the cell by displacing it from the plasma membrane through binding to the DID-DD region of mDia. Here, we review these findings and discuss how Liprin-α regulates the Rho-mDia pathway and how the mDia-Liprin-α complex functions in vivo.
Liprin; Rho; actin cytoskeleton; formin; mDia
The radial spoke (RS) is a complex of at least 23 proteins that works as a mechanochemical transducer between the central‐pair apparatus and the peripheral microtubule doublets in eukaryotic flagella and motile cilia. The RS contributes to the regulation of the activity of dynein motors, and thus to flagellar motility. Despite numerous biochemical, physiological and structural studies, the mechanism of the function of the radial spoke remains unclear. Detailed knowledge of the 3D structure of the RS protein complex is needed in order to understand how RS regulates dynein activity. Here we review the most important findings on the structure of the RS, including results of our recent cryo‐electron tomographic analysis of the RS protein complex.
axoneme; cilia; cryo‐electron tomography; dynein; flagella; motility; radial spokes
SNARE complexes mediate membrane fusion in the endomembrane system. They consist of coiled-coil bundles of four helices designated as Qa, Qb, Qc and R. A critical intermediate in the fusion pathway is the trans-SNARE complex generated by the assembly of SNAREs residing in opposing membranes. Mechanistic details of trans-SNARE complex formation and topology in a physiological system remain largely unresolved. Our studies on native yeast vacuoles revealed that SNAREs alone are insufficient to form trans-SNARE complexes and that additional factors, potentially tethering complexes and Rab GTPases, are required for the process. Here we report a novel finding that a HOPS tethering complex dimer catalyzes Rab GTPase-dependent formation of a topologically preferred QbQcR-Qa trans-SNARE complex.
HOPS tethering complex dimer; QbQcR-Qa trans-SNARE complex; Rab GTPase
Kinesin-5 mechanoenzymes drive mitotic spindle dynamics as slow, processive microtubule (MT)-plus-end directed motors. Surprisingly, the Saccharomyces cerevisiae kinesin-5 Cin8 was recently found to be bi-directional: it can move processively in both directions on MTs. Two hypotheses have been suggested for the mechanism of the directionality switch: (1) single molecules of Cin8 are intrinsically minus-end directed, but mechanical coupling between two or more motors triggers the switch; (2) a single motor can switch direction, and “cargo binding” i.e., binding between two MTs triggers the switch to plus-end motility. Single-molecule fluorescence data we published recently, and augment here, favor hypothesis (2). In low-ionic-strength conditions, single molecules of Cin8 move in both minus- and plus-end directions. Fluorescence photo bleaching data rule out aggregation of Cin8 while they move in the plus and in the minus direction. The evidence thus points toward cargo regulation of directionality, which is likely to be related to cargo regulation in other kinesins. The molecular mechanisms of this regulation, however, remain to be elucidated.
Saccharomyces cerevisiae Cin8; kinesin directionality; kinesin-5; microtubules; mitosis
Volume 1 has defined the scope of BioArchitecture. From the outset we have strived to ensure that BioArchitecture is not limited to the three major polymer systems of the cytoplasm. I am happy to say that a cursory glance at the contents of volume 1 makes it clear that we are interested in all aspects of bioarchitecture from molecules to polymers to cells to tissue to the organism.
Actin polymerization plays a major role in many cellular processes, including cell motility, vesicle trafficking, and pathogen propulsion. The transformation of the (protrusive) polymerization forces into directed motion requires that the growing filaments are positioned next to the surface. This is achieved by localization of surface actin nucleators (WASP), which then activate Arp2/3 complex to form new actin branches. Yet, the same surface-bound WASP molecule which initiates the nucleation of new actin branches, also inherently prevents the translation of the polymerization forces into motion, essentially because the WASP molecule has to be in contact with the network during the formation of the new branch. In our recent paper we show that cortactin relaxes this internal inhibition by enhancing the release of WASP-VCA molecule from the new branching site after nucleation is initiated. We show that this enhanced release has two major effects; it increases the turnover rate of branching per WASP molecule, and it decreases the friction-like force caused by the binding of the moving surface with respect to the growing actin network.
Arp2/3 complex; WASP-VCA; actin-based motility; cortactin; friction-like force; propulsion velocity
Chemotaxis is crucial for many physiological processes including the recruitment of leukocytes to sites of infection, trafficking of lymphocytes in the human body, and metastasis of cancer cells. A family of small proteins, chemokines, serves as the signals, and a family of G-protein coupled receptors (GPCRs) detects chemokines and direct cell migration. One of the basic questions in chemotaxis of eukaryotes is how a GPCR transduces signals to control the assembly of the actin network that generates directional force for cell migration. Over the past decade, a variety of signaling components have been implicated to transduce the GPCR signaling to the actin cytoskeleton. Studies in a lower eukaryotic organism, Dictyostelium discoideum, have allowed us to discover evolutionary conversed components involved in the GPCR-controlled actin network during chemotaxis. However, complete pathways linking GPCR to the actin network are still far from clear. Here we first summarize the previous studies on these components, and then update with our finding showing a new pathway, consisting of a GPCR, Gβγ, Elmo/Dock, Rac and Arp2/3 and actin. We suggest that this pathway serves as a direct linkage between the GPCR/G-protein, the chemoattractant sensing machinery, and the actin cytoskeleton, the machinery of cell movement during chemotaxis of eukaryotic cells.
Dictyostelium; Dock; Elmo; GPCR; actin; chemotaxis; cytoskeleton; signaling