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1.  Hook1, microtubules, and Rab22 
Bioarchitecture  2013;3(5):141-146.
Clathrin-independent endocytosis (CIE) mediates the internalization of many plasma membrane (PM) proteins involved in homeostasis, immune response, and signaling. CIE cargo molecules are internalized independent of clathrin, and dynamin, and modulated by the small G protein Arf6. After internalization the CIE cargo proteins either follow a default pathway of trafficking to lysosomes for degradation or follow a pathway where they are routed directly to the recycling endosomes for return to the PM. The selective endosomal sorting of molecules like CD44, CD98, and CD147, which are involved in cell-cell and cell-extracellular interactions, indicates that sorting mechanisms dictate the post-endocytic fate of CIE cargo proteins. In a recent study, we identified sorting signals that specify the endosomal trafficking of CIE cargo proteins and uncover a role for Hook1 as an endosomal cargo adaptor that routes CIE cargo to the recycling endosomes. Furthermore, we found that Hook1, microtubules, and Rab22a work in coordination to directly recycle the cargo and facilitate cell spreading. Here, we discuss our current view on the endosomal sorting of CIE cargo proteins and their molecular regulators.
doi:10.4161/bioa.26638
PMCID: PMC3907461  PMID: 24284901
clathrin-independent endocytosis; Hook1; Rab22a; Rab22; microtubules; endosomal sorting; sorting signals; recycling; basigin; CD147
2.  Periodicities designed in the tropomyosin sequence and structure define its functions 
Bioarchitecture  2013;3(3):51-56.
Tropomyosin is an actin binding protein that regulates actin filament dynamics and its interactions with actin binding proteins such as myosin, tropomodulin, formin, Arp2/3 and ADF-cofilin in most eukaryotic cells. Tropomyosin is the prototypical two-chained, α-helical coiled coil protein that associates end-to-end and binds to both sides of the actin filament. Each tropomyosin molecule spans four to seven actin monomers in the filament, depending on the size of the tropomyosin. Tropomyosins have a periodic heptad repeat sequence that is characteristic of coiled coil proteins as well as additional periodicities required for its interaction with the actin filament, where each periodic repeat interacts with one actin molecule. This review addresses the role of periodic features of the Tm molecule in carrying out its universal functions of binding to the actin filament and its regulation and the specific features that may determine the isoform specificity of tropomyosins.
doi:10.4161/bioa.25616
PMCID: PMC3782539  PMID: 23887197
tropomyosin; muscle regulation; actin filament; cytoskeleton; coiled coil
3.  The vacuole within 
Bioarchitecture  2013;3(3):64-68.
The notochord is an evolutionarily conserved structure that has long been known to play an important role in patterning during embryogenesis. Structurally, the notochord is composed of two cell layers: an outer epithelial-like sheath, and an inner core of cells that contain large fluid-filled vacuoles. We have recently shown these notochord vacuoles are lysosome-related organelles that form through Rab32a and vacuolar-type proton-ATPase-dependent acidification. Disruption of notochord vacuoles results in a shortened embryo along the anterior-posterior axis. Interestingly, we discovered that notochord vacuoles are also essential for proper spine morphogenesis and that vacuole defects lead to scoliosis of the spine. Here we discuss the cellular organization of the notochord and how key features of its architecture allow the notochord to function in embryonic axis elongation and spine formation.
doi:10.4161/bioa.25503
PMCID: PMC3782541  PMID: 23887209
zebrafish; notochord vacuole; lysosome-related organelle; axis elongation; spine formation; scoliosis
4.  New insights on vertebrate olivo-cerebellar climbing fibers from computerized morphological reconstructions 
Bioarchitecture  2013;3(2):38-41.
Characterization of neuronal connectivity is essential to understanding the architecture of the animal nervous system. Specific labeling and imaging techniques can visualize axons and dendrites of single nerve cells. Two-dimensional manual drawing has long been used to describe the morphology of labeled neuronal elements. However, quantitative morphometry, which is essential to understanding functional significance, cannot be readily extracted unless the detailed neuronal geometry is comprehensively reconstructed in three-dimensional space. We have recently applied an accurate and robust digital reconstruction system to cerebellar climbing fibers, which form highly dense and complex terminal arbors as one of the strongest presynaptic endings in the vertebrate nervous system. Resulting statistical analysis has shown how climbing fibers morphology is special in comparison to other axonal terminals. While thick primary branches may convey excitation quickly and faithfully to the far ends, thin tendril branches, which have a larger bouton density, form the majority of presynaptic outputs. This data set, now publicly available from NeuroMorpho.Org for further modeling and analysis, may constitute the first detailed and comprehensive digital reconstruction of the complete axonal terminal field with identified branch types and full accounting of boutons for any neuronal class in the vertebrate brain.
doi:10.4161/bioa.24062
PMCID: PMC3715541  PMID: 23756373
axon; terminal arbor; bouton; branches; tendril; cerebellar cortex; molecular layer; biotinylated dextran amine; rat
5.  Dickkopf-3 function in the prostate 
Bioarchitecture  2013;3(2):42-44.
The tumor suppressor Dickkopf-3 (Dkk-3) is rather a unique molecule. Although it is related to the Dickkopf family of secreted Wnt antagonists, it does not directly inhibit Wnt signaling, and its function and mechanism of action are unknown. Endogenous Dkk-3 was recently found to be required to limit cell proliferation both in the developing mouse prostate and in 3D cultures of human prostate epithelial cells. Dkk-3 was further shown to modulate the response of normal prostate epithelial cells to transforming growth factor-β (TGF-β). These studies are consistent with a model in which Dkk-3 is required by normal cells to prevent the TGF-β switch from tumor suppressor to tumor promoter. Here, we discuss these findings and their potential impact on the development and progression of prostate cancer.
doi:10.4161/bioa.25243
PMCID: PMC3715542  PMID: 23765605
Dkk-3; TGF-β; acinar morphogenesis; prostate
6.  Supracellular actomyosin assemblies during development 
Bioarchitecture  2013;3(2):45-49.
Changes in cell shape are one of the driving forces of tissue morphogenesis. Contractile cytoskeletal assemblies based on actomyosin networks have emerged as a main player that can drive these changes. Different types of actomyosin networks have been identified, with distinct subcellular localizations, including apical junctional and apicomedial actomyosin. A further specialization of junctional actomyosin are so-called actomyosin ‘cables’, supracellular arrangements that appear to stretch over many cell diameters. Such actomyosin cables have been shown to serve several important functions, in processes such as wound healing, epithelial morphogenesis and maintenance of compartment identities during development. In the Drosophila embryo, we have recently identified a function for a circumferential actomyosin cable in assisting tube formation. Here, I will briefly summarize general principles that have emerged from the analysis of such cables.
doi:10.4161/bioa.25339
PMCID: PMC3715543  PMID: 23760352
Actomyosin; morphogenesis; cable; wound healing; Drosophila; anisotropy; development
7.  Human fronto-parietal and parieto-hippocampal pathways represent behavioral priorities in multiple spatial reference frames 
Bioarchitecture  2013;3(5):147-152.
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.
doi:10.4161/bioa.27462
PMCID: PMC3907462  PMID: 24322829
parietal cortex; frontal cortex; spatial attention; coordinate transformation; connectivity; saccade; grasp; reach; navigation
8.  Axonal trafficking of NMNAT2 and its roles in axon growth and survival in vivo 
Bioarchitecture  2013;3(5):133-140.
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.
doi:10.4161/bioa.27049
PMCID: PMC3907460  PMID: 24284888
NMNAT2; Wallerian degeneration; axon growth; axon survival; axonal transport; neurodegeneration; palmitoylation; ubiquitin proteasome
9.  Integration of signaling and cytoskeletal remodeling by Nck in directional cell migration 
Bioarchitecture  2013;3(3):57-63.
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.
doi:10.4161/bioa.25744
PMCID: PMC3782540  PMID: 23887203
Nck; actin polymerization; cell migration; cell polarity; adhesion dynamics; WASp; morphogenesis; endothelial cell
10.  Insights from an erroneous kinetochore-microtubule attachment state 
Bioarchitecture  2013;3(3):69-76.
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.
doi:10.4161/bioa.25734
PMCID: PMC3782542  PMID: 23887229
cell division; kinetochore; microtubule; syntelic attachment; error correction; spindle assembly checkpoint; aurora B kinase; Mad1; BubR1
11.  Aged skeletal muscle retains the ability to fully regenerate functional architecture 
Bioarchitecture  2013;3(2):25-37.
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.
doi:10.4161/bioa.24966
PMCID: PMC3715540  PMID: 23807088
skeletal muscle; aging; progenitor cells; fiber branching; contractility
12.  Vacuoles in mammals 
Bioarchitecture  2013;3(1):13-19.
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.
doi:10.4161/bioa.24126
PMCID: PMC3639239  PMID: 23572040
vacuole; endocytosis; embryogenesis; rab7; Vam2/Vps41
13.  Welcome to BioArchitecture 2013 
Bioarchitecture  2013;3(1):1.
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.
doi:10.4161/bioa.24027
PMCID: PMC3639238
14.  Rotary ATPases 
Bioarchitecture  2013;3(1):2-12.
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.
doi:10.4161/bioa.23301
PMCID: PMC3639240  PMID: 23369889
biological motors; rotary motors; energy conversion; ATP synthase; vacuolar ATPase; A-type ATPase; structural biology; X-ray crystallography; electron microscopy
15.  Spatiotemporal regulation of meiotic recombination by Liaisonin 
Bioarchitecture  2013;3(1):20-24.
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.
doi:10.4161/bioa.23966
PMCID: PMC3639241  PMID: 23572041
Meiotic recombination; Spo11; DNA double-strand break formation; higher-order chromosome structure; S phase checkpoint
16.  Sizing up long non-coding RNAs 
Bioarchitecture  2012;2(6):189-199.
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.
doi:10.4161/bioa.22592
PMCID: PMC3527312  PMID: 23267412
RNA; non-coding; long noncoding RNA; lncRNA; lincRNA; epigenetics; cancer; hormone receptor; secondary structure; structural biology; RNA structure; HOTAIR; MALAT
17.  BioArchitecture 
Bioarchitecture  2012;2(6):200-203.
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.
doi:10.4161/bioa.22726
PMCID: PMC3527313  PMID: 23267413
actin; cytoskeleton; microtubules; intermediate filaments; nuclear structure; protein folding; isoform sorting
18.  The role of the cofilin-actin rod stress response in neurodegenerative diseases uncovers potential new drug targets 
Bioarchitecture  2012;2(6):204-208.
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.
doi:10.4161/bioa.22549
PMCID: PMC3527314  PMID: 23267414
Huntington disease; actin; cofilin; cofilin rods; cytoskeleton; nuclear transport signals; profilin
19.  Akirin 
Bioarchitecture  2012;2(6):209-213.
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.
doi:10.4161/bioa.22907
PMCID: PMC3527315  PMID: 23242134
Akirin; Twist; SWI/SNF; chromatin; transcription; muscle; Drosophila
20.  Fibroblast growth factor receptor 3 regulates microtubule formation and cell surface mechanical properties in the developing organ of Corti 
Bioarchitecture  2012;2(6):214-219.
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.
doi:10.4161/bioa.22332
PMCID: PMC3527316  PMID: 23267415
Fibroblast growth factor; Young’s modulus; hair cell; pillar cell
21.  Microtubule dynamics regulation contributes to endothelial morphogenesis 
Bioarchitecture  2012;2(6):220-227.
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.
doi:10.4161/bioa.22335
PMCID: PMC3527317  PMID: 23267416
CLASP2; blebbistatin; cell migration; cytoskeleton; endothelial cells; in vitro angiogenesis; microtubule dynamics; nocodazole
22.  Intravital microscopy 
Bioarchitecture  2012;2(5):143-157.
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.
doi:10.4161/bioa.21758
PMCID: PMC3696059  PMID: 22992750
23.  Myosins in cell junctions 
Bioarchitecture  2012;2(5):158-170.
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.
doi:10.4161/bioa.21791
PMCID: PMC3696060  PMID: 22954512
Myo10; Myo15a; Myo1e; Myo6; Myo7a; Myo9a; Myo9b; adherens junction; dachs; myosin; nonmuscle myosin; tight junction
24.  Competition and compensation 
Bioarchitecture  2012;2(5):171-174.
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.
doi:10.4161/bioa.21733
PMCID: PMC3696061  PMID: 22954581
myosin; actin; filopodia; cytoskeleton; motor proteins; stereocilia; deafness
25.  Tethering factor P115 
Bioarchitecture  2012;2(5):175-180.
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.
doi:10.4161/bioa.21702
PMCID: PMC3696062  PMID: 22992751
p115; SNARE; tethering; Golgi; coiled-coil domain

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