We represent behaviorally relevant information in different spatial reference frames in order to interact effectively with our environment. For example, we need an egocentric (e.g., body-centered) reference frame to specify limb movements and an allocentric (e.g., world-centered) reference frame to navigate from one location to another. Posterior parietal cortex (PPC) is vital for performing transformations between these different coordinate systems. Here, we review evidence for multiple pathways in the human brain, from PPC to motor, premotor, and supplementary motor areas, as well as to structures in the medial temporal lobe. These connections are important for transformations between egocentric reference frames to facilitate sensory-guided action, or from egocentric to allocentric reference frames to facilitate spatial navigation.
parietal cortex; frontal cortex; spatial attention; coordinate transformation; connectivity; saccade; grasp; reach; navigation
The NAD-synthesizing enzyme NMNAT2 is critical for axon survival in primary culture and its depletion may contribute to axon degeneration in a variety of neurodegenerative disorders. Here we discuss several recent reports from our laboratory that establish a critical role for NMNAT2 in axon growth in vivo in mice and shed light on the delivery and turnover of this survival factor in axons. In the absence of NMNAT2, axons fail to extend more than a short distance beyond the cell body during embryonic development, implying a requirement for NMNAT2 in axon maintenance even during development. Furthermore, we highlight findings regarding the bidirectional trafficking of NMNAT2 in axons on a vesicle population that undergoes fast axonal transport in primary culture neurites and in mouse sciatic nerve axons in vivo. Surprisingly, loss of vesicle association boosts the axon protective capacity of NMNAT2, an effect that is at least partially mediated by a longer protein half-life of cytosolic NMNAT2 variants. Analysis of wild-type and variant NMNAT2 in mouse sciatic nerves and Drosophila olfactory receptor neuron axons supports the existence of a similar mechanism in vivo, highlighting the potential for regulation of NMNAT2 stability and turnover as a mechanism to modulate axon degeneration in vivo.
NMNAT2; Wallerian degeneration; axon growth; axon survival; axonal transport; neurodegeneration; palmitoylation; ubiquitin proteasome
Planar and apical-basal cellular polarization of epithelia and endothelia are crucial during morphogenesis. The establishment of these distinct polarity states and their transitions are regulated by signaling networks that include polarity complexes, Rho GTPases, and phosphoinositides. The spatiotemporal coordination of signaling by these molecules modulates cytoskeletal remodeling and vesicle trafficking to specify membrane domains, a prerequisite for the organization of tissues and organs. Here we present an overview of how activation of the WASp/Arp2/3 pathway of actin remodeling by Nck coordinates directional cell migration and speculate on its role as a signaling integrator in the coordination of cellular processes involved in endothelial cell polarity and vascular lumen formation.
Nck; actin polymerization; cell migration; cell polarity; adhesion dynamics; WASp; morphogenesis; endothelial cell
Faithful distribution of the genome requires that sister kinetochores, which assemble on each chromatid during cell division, interact with dynamic microtubules from opposite spindle poles in a configuration called chromosome biorientation. Biorientation produces tension that increases the affinity of kinetochores for microtubules via ill-defined mechanisms. Non-bioriented kinetochore-microtubule (kt-MT) interactions are prevalent but short-lived due to an error correction pathway that reduces the affinity of kinetochores for microtubules. Interestingly, incorrect kt-MT interactions can be stabilized by experimentally applying force to misoriented chromosomes. Here, a live-cell force assay is utilized to characterize the molecular composition of a common type of improper kt-MT attachment. Our force-related studies are also discussed in the context of current models for tension-dependent stabilization of kt-MT interactions.
cell division; kinetochore; microtubule; syntelic attachment; error correction; spindle assembly checkpoint; aurora B kinase; Mad1; BubR1
While the general understanding of muscle regenerative capacity is that it declines with increasing age due to impairments in the number of muscle progenitor cells and interaction with their niche, studies vary in their model of choice, indices of myogenic repair, muscle of interest and duration of studies. We focused on the net outcome of regeneration, functional architecture, compared across three models of acute muscle injury to test the hypothesis that satellite cells maintain their capacity for effective myogenic regeneration with age. Muscle regeneration in extensor digitorum longus muscle (EDL) of young (3 mo-old), old (22 mo-old) and senescent female mice (28 mo-old) was evaluated for architectural features, fiber number and central nucleation, weight, collagen and fat deposition. The 3 injury paradigms were: a myotoxin (notexin) which leaves the blood vessels and nerves intact, freezing (FI) that damages local muscle, nerve and blood vessels and denervation-devascularization (DD) which dissociates the nerves and blood vessels from the whole muscle. Histological analyses revealed successful architectural regeneration following notexin injury with negligible fibrosis and fully restored function, regardless of age. In comparison, the regenerative response to injuries that damaged the neurovascular supply (FI and DD) was less effective, but similar across the ages. The focus on net regenerative outcome demonstrated that old and senescent muscle has a robust capacity to regenerate functional architecture.
skeletal muscle; aging; progenitor cells; fiber branching; contractility
A vacuole is a membrane-bound subcellular structure involved in intracellular digestion. Instead of the large “vacuolar” organelles that are found in plants and fungi, animal cells possess lysosomes that are smaller in size and are enriched with hydrolytic enzymes similar to those found in the vacuoles. Large vacuolar structures are often observed in highly differentiated mammalian tissues such as embryonic visceral endoderm and absorbing epithelium. Vacuoles/lysosomes share a conserved mechanism of biogenesis, and they are at the terminal of the endocytic pathways, Recent genetic studies of the mammalian orthologs of Vam/Vps genes, which have essential functions for vacuole assembly, revealed that the dynamics of vacuoles/lysosomes are important for tissue differentiation and patterning through regulation of various molecular signaling events in mammals.
vacuole; endocytosis; embryogenesis; rab7; Vam2/Vps41
The first major hurdle for any new journal is to achieve acceptance into Medline/PubMed. I am pleased to report that BioArchitecture was accepted into Medline/PubMed in August 2012. This means that accepted manuscripts are immediately visible through their listing on PubMed. This is very welcome news to contributors to the journal and makes the journal a more attractive destination for publication of new research findings.
Rotary ATPases are molecular rotary motors involved in biological energy conversion. They either synthesize or hydrolyze the universal biological energy carrier adenosine triphosphate. Recent work has elucidated the general architecture and subunit compositions of all three sub-types of rotary ATPases. Composite models of the intact F-, V- and A-type ATPases have been constructed by fitting high-resolution X-ray structures of individual subunits or sub-complexes into low-resolution electron densities of the intact enzymes derived from electron cryo-microscopy. Electron cryo-tomography has provided new insights into the supra-molecular arrangement of eukaryotic ATP synthases within mitochondria and mass-spectrometry has started to identify specifically bound lipids presumed to be essential for function. Taken together these molecular snapshots show that nano-scale rotary engines have much in common with basic design principles of man made machines from the function of individual “machine elements” to the requirement of the right “fuel” and “oil” for different types of motors.
biological motors; rotary motors; energy conversion; ATP synthase; vacuolar ATPase; A-type ATPase; structural biology; X-ray crystallography; electron microscopy
Sexual reproduction involves diversification of genetic information in successive generations. Meiotic recombination, which substantially contributes to the increase in genetic diversity, is initiated by programmed DNA double-strand breaks (DSBs) catalyzed by the evolutionarily conserved Spo11 protein. Spo11 requires additional partner proteins for its DNA cleavage reaction. DSBs are preferentially introduced at defined chromosomal sites called “recombination hotspots.” Recent studies have revealed that meiotically established higher-order chromosome structures, such as chromosome axes and loops, are also crucial in the control of DSB formation. Most of the DSB sites are located within chromatin loop regions, while many of the proteins involved in DSB formation reside on chromosomal axes. Hence, DSB proteins and DSB sites seem to be distantly located.
To resolve this paradox, we conducted comprehensive proteomics and ChIP-chip analyses on Spo11 partners in Schizosaccharomyces pombe, in combination with mutant studies. We identified two distinct DSB complexes, the “DSBC (DSB Catalytic core)“ and “SFT (Seven-Fifteen-Twenty four; Rec7-Rec15-Rec24)” subcomplexes. The DSBC subcomplex contains Spo11 and functions as the catalytic core for the DNA cleavage reaction. The SFT subcomplex is assumed to execute regulatory functions. To activate the DSBC subcomplex, the SFT subcomplex tethers hotspots to axes via its interaction with Mde2, which can interact with proteins in both DSBC and SFT subcomplexes. Thus, Mde2 is likely to bridge these two subcomplexes, forming a “tethered loop-axis complex.” It should be noted that Mde2 expression is strictly regulated by S phase checkpoint monitoring of the completion of DNA replication. From these observations, we proposed that Mde2 is a central coupler for meiotic recombination initiation to establish a tethered loop-axis complex in liaison with the S phase checkpoint.
Meiotic recombination; Spo11; DNA double-strand break formation; higher-order chromosome structure; S phase checkpoint
Long noncoding RNAs (lncRNAs) play a key role in many important areas of epigenetics, stem cell biology, cancer, signaling and brain function. This emerging class of RNAs constitutes a large fraction of the transcriptome, with thousands of new lncRNAs reported each year. The molecular mechanisms of these RNAs are not well understood. Currently, very little structural data exist. We review the available lncRNA sequence and secondary structure data. Since almost no tertiary information is available for lncRNAs, we review crystallographic structures for other RNA systems and discuss the possibilities for lncRNAs in the context of existing constraints.
RNA; non-coding; long noncoding RNA; lncRNA; lincRNA; epigenetics; cancer; hormone receptor; secondary structure; structural biology; RNA structure; HOTAIR; MALAT
BioArchitecture is a term used to describe the organization and regulation of biological space. It applies to the principles which govern the structure of molecules, polymers and mutiprotein complexes, organelles, membranes and their organization in the cytoplasm and the nucleus. It also covers the integration of cells into their three dimensional environment at the level of cell-matrix, cell-cell interactions, integration into tissue/organ structure and function and finally into the structure of the organism. This review will highlight studies at all these levels which are providing a new way to think about the relationship between the organization of biological space and the function of biological systems.
actin; cytoskeleton; microtubules; intermediate filaments; nuclear structure; protein folding; isoform sorting
The cofilin-actin rod stress response is an actin cytoskeletal dynamic arrest that occurs in cells under a variety of stress conditions. Upon stress, the rapidly activated cofilin saturates actin filaments causing them to bundle into rod structures in either the nucleus or cytoplasm, halting actin polymerization and thus freeing ATP. Importantly, these rods dissociate quickly following relief of the transient stress. The rods form inappropriately in neurons involved in the progression of Alzheimer disease (AD) and we have linked dysfunctional dynamics of the nuclear rod response to Huntington disease (HD). Cofilin levels are also perturbed in Parkinson disease (PD), and profilin, an actin binding protein with opposite action to cofilin, is mutated in Amyotrophic Lateral Sclerosis (ALS). The persistence of the rods post-stress suggests that critical molecular switches to turn this response both on and off are being affected in neurodegeneration. We have recently shown that the cofilin protein is regulated by highly conserved nuclear import and export signals and that these signals are required to be functional for an appropriate rod formation during stress. The ability of cofilin to form rods is required in a cell culture model for cells to be resistant to apoptosis under stress conditions, indicating that a normal cofilin-actin rod response is likely integral to proper cell health in higher order organisms. Here we hypothesize on the potential physiological function of nuclear cofilin-actin rods and why the dysregulation of this response could lead to the selective vulnerability of the most susceptible populations of cells in HD. We further suggest that learning more about this cytoskeletal cell stress response will open up new avenues for drug target discovery in neurodegenerative disorders.
Huntington disease; actin; cofilin; cofilin rods; cytoskeleton; nuclear transport signals; profilin
Embryonic patterning relies upon an exquisitely timed program of gene regulation. While the regulation of this process via the action of transcription factor networks is well understood, new lines of study have highlighted the importance of a concurrently regulated program of chromatin remodeling during development. Chromatin remodeling refers to the manipulation of the chromatin architecture through rearrangement, repositioning, or restructuring of nucleosomes to either favor or hinder the expression of associated genes. While the role of chromatin remodeling pathways during tumor development and cancer progression are beginning to be clarified, the roles of these pathways in the course of tissue specification, morphogenesis and patterning remains relatively unknown. Further, relatively little is understood as to the mechanism whereby developmentally critical transcription factors coordinate with chromatin remodeling factors to optimize target gene loci for gene expression. Such a mechanism might involve direct transcription factor/chromatin remodeling factor interactions, or could likely be mediated via an unknown intermediary. Our group has identified the relatively unknown protein Akirin as a putative member of this latter group: a secondary cofactor that serves as an interface between a developmentally critical transcription factor and the chromatin remodeling machinery. This role for the Akirin protein suggests a novel regulatory mode for regulating gene expression during development.
Akirin; Twist; SWI/SNF; chromatin; transcription; muscle; Drosophila
Fibroblast Growth Factor (Fgf) signaling is involved in the exquisite cellular patterning of the developing cochlea, and is necessary for proper hearing function. Our previous data indicate that Fgf signaling disrupts actin, which impacts the surface stiffness of sensory outer hair cells (OHCs) and non-sensory supporting pillar cells (PCs) in the organ of Corti. Here, we used Atomic Force Microscopy (AFM) to measure the impact of loss of function of Fgf-receptor 3, on cytoskeletal formation and cell surface mechanical properties. We find a 50% decrease in both OHC and PC surface stiffness, and a substantial disruption in microtubule formation in PCs. Moreover, we find no change in OHC electromotility of Fgfr3-deficient mice. To further understand the regulation by Fgf-signaling on microtubule formation, we treated wild-type cochlear explants with Fgf-receptor agonist Fgf2, or antagonist SU5402, and find that both treatments lead to a significant reduction in β-Tubulin isotypes I&II. To identify downstream transcriptional targets of Fgf-signaling, we used QPCR arrays to probe 84 cytoskeletal regulators. Of the 5 genes significantly upregulated following treatment, Clasp2, Mapre2 and Mark2 impact microtubule formation. We conclude that microtubule formation is a major downstream effector of Fgf-receptor 3, and suggest this pathway impacts the formation of fluid spaces in the organ of Corti.
Fibroblast growth factor; Young’s modulus; hair cell; pillar cell
Because little is known how microtubules contribute to cell migration in a physiological three-dimensional environment, we analyzed microtubule function and dynamics during in vitro angiogenesis in which endothelial cells form networks on a reconstituted basement membrane. Endothelial network formation resulted from distinct cell behaviors: matrix reorganization by myosin-mediated contractile forces, and active cell migration along reorganized, bundled matrix fibers. Inhibition of microtubule dynamics inhibited persistent cell migration, but not matrix reorganization. In addition, microtubule polymerization dynamics and CLASP2-binding to microtubules were spatially regulated to promote microtubule growth into endothelial cell protrusions along matrix tension tracks. We propose that microtubules counter-act contractile forces of the cortical actin cytoskeleton and are required to stabilize endothelial cell protrusions in a soft three-dimensional environment.
CLASP2; blebbistatin; cell migration; cytoskeleton; endothelial cells; in vitro angiogenesis; microtubule dynamics; nocodazole
Intravital microscopy is an extremely powerful tool that enables imaging several biological processes in live animals. Recently, the ability to image subcellular structures in several organs combined with the development of sophisticated genetic tools has made possible extending this approach to investigate several aspects of cell biology. Here we provide a general overview of intravital microscopy with the goal of highlighting its potential and challenges. Specifically, this review is geared toward researchers that are new to intravital microscopy and focuses on practical aspects of carrying out imaging in live animals. Here we share the know-how that comes from first-hand experience, including topics such as choosing the right imaging platform and modality, surgery and stabilization techniques, anesthesia and temperature control. Moreover, we highlight some of the approaches that facilitate subcellular imaging in live animals by providing numerous examples of imaging selected organelles and the actin cytoskeleton in multiple organs.
The development of cell-cell junctions was a fundamental step in metazoan evolution, and human health depends on the formation and function of cell junctions. Although it has long been known that actin and conventional myosin have important roles in cell junctions, research has begun to reveal the specific functions of the different forms of conventional myosin. Exciting new data also reveals that a growing number of unconventional myosins have important roles in cell junctions. Experiments showing that cell junctions act as mechanosensors have also provided new impetus to understand the functions of myosins and the forces they exert. In this review we will summarize recent developments on the roles of myosins in cell junctions.
Myo10; Myo15a; Myo1e; Myo6; Myo7a; Myo9a; Myo9b; adherens junction; dachs; myosin; nonmuscle myosin; tight junction
Stereocilia are actin protrusions with remarkably well-defined lengths and organization. A flurry of recent papers has reported multiple myosin motor proteins involved in regulating stereocilia structures by transporting actin-regulatory cargo to the tips of stereocilia.1-13 In our recent paper, we show that two paralogous class 3 myosins — Myo3a and Myo3b — both transport the actin-regulatory protein Espin 1 (Esp1) to stereocilia and filopodia tips in a remarkably similar, albeit non-identical fashion.1 Here we present experimental and computational data that suggests that subtle differences between these two proteins’ biophysical and biochemical properties can help us understand how these myosin species target and regulate the lengths of actin protrusions.
myosin; actin; filopodia; cytoskeleton; motor proteins; stereocilia; deafness
The membrane tethering factor p115 has been shown to have important functions in ER to Golgi traffic and Golgi biogenesis. The multidomain structure of p115 allows for interactions with a diverse array of proteins that govern cargo movement at the ER-Golgi interface. Within its C-terminal region p115 contains four coiled-coil domains (CC1-CC4). Of the four coiled-coils, only CC1 has been shown to be required for p115 function, presumably by its ability to bind numerous SNARE proteins as well as the small GTPase Rab1. Recently, we showed that CC4 also interacts with SNARE proteins and that CC4 is required for p115 function in Golgi homeostasis and the trafficking of transmembrane but not soluble cargo. Here, we propose a novel model wherein p115 facilitates membrane tethering and fusion by simultaneously engaging its CC1 and CC4 domains with distinct SNARE proteins to promote formation of SNARE complexes.
p115; SNARE; tethering; Golgi; coiled-coil domain
Growth factors and their receptors are important for cellular migration as well as axonal guidance and myelination in the brain. They also play a key role in programmed cell death, and are implicated in a number of mental illnesses. Recently, we reported that healthy young adults who carry the T allele variant in the growth factor gene, NTRK1 (at location rs6336), had lower white matter integrity than non-carriers on diffusion images of the brain. Diffusion tensor imaging (DTI) revealed how this single nucleotide polymorphism affects white matter microstructure in human populations; DTI is also used to identify characteristic features of brain connectivity in typically developing children and in patients. Newly discovered links between neuroimaging measures and growth factors whose molecular neuroscience is well known offer an important step in understanding mechanisms that contribute to brain connectivity. Altered fiber connectivity may mediate the relationship between some genetic risk factors and a variety of mental illnesses.
neurotrophin; growth factor; tropomyosin-related kinase receptor A; neurotrophic tyrosine kinase receptor 1; myelin; development; fractional anisotropy; radial diffusivity; diffusion tensor imaging; schizophrenia
We recently identified the atypical myosin, Myosin VI, as a component of epithelial cell-cell junctions that interacts with E-cadherin. Recombinant proteins bearing the cargo-binding domain of Myosin VI (Myo VI-CBD) or the cytoplasmic tail of E-cadherin can interact directly with one another. In this report we further investigate the molecular requirements of the interaction between Myo VI-CBD and E-cadherin combining truncation mutation analysis with in vitro binding assays. We report that a short (28 amino acid) juxtamembrane region of the cadherin cytoplasmic tail is sufficient to bind Myo VI-CBD. However, central regions of the cadherin tail adjacent to the juxtamembrane sequence also display binding activity for Myo VI-CBD. It is therefore possible that the cadherin tail bears two binding sites for Myosin VI, or an extended binding site that includes the juxtamembrane region. Nevertheless, our biochemical data highlight the capacity for the juxtamembrane region to interact with functionally-significant cytoplasmic proteins.
E-cadherin; myosin VI; cytoskeleton; protein-protein interaction; Epithelia
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