Tension sensing of bi-oriented chromosomes is essential for the fidelity of chromosome segregation. The spindle assembly checkpoint (SAC) conveys lack of tension or attachment to the anaphase promoting complex. Components of the SAC (Bub1) phosphorylate histone H2A (S121) and recruit the protector of cohesin, Shugoshin (Sgo1) to the inner centromere. How the chromatin structural modifications of the inner centromere are integrated into the tension sensing mechanisms and the checkpoint are not known.
We have identified a Bub1/Sgo1 dependent structural change in the geometry and dynamics of kinetochores and the pericentric chromatin upon reduction of microtubule dynamics. The cluster of inner kinetochores contract while the pericentric chromatin and cohesin that encircle spindle microtubules undergo a radial expansion. Despite its increased spatial distribution, the pericentric chromatin is less dynamic. The change in dynamics is due to histone H2A phosphorylation and Sgo1 recruitment to the pericentric chromatin, rather than microtubule dynamics.
Bub1 and Sgo1 act as a rheostat to regulate the chromatin spring and maintain force balance. Through Histone H2A S121 phosphorylation and recruitment of Sgo1, Bub1 kinase softens the chromatin spring in response to changes in microtubule dynamics. The geometric alteration of all 16 kinetochores and pericentric chromatin reflect global changes in the pericentromeric region and provide mechanisms for mechanically amplifying damage at a single kinetochore microtubule.
Neurite formation is a seminal event in the early development of neurons. However, little is known about the mechanisms by which neurons form neurites. F-BAR proteins function in sensing and inducing membrane curvature [1, 2]. Cdc42-interacting protein 4 (CIP4), a member of the F-BAR family, regulates endocytosis in a variety of cell types [3–9]. However, there is little data on how CIP4 functions in neurons [10, 11]. Here we show that CIP4 plays a novel role in neuronal development by inhibiting neurite formation. Remarkably, CIP4 exerts this effect not through endocytosis, but by producing lamellipodial protrusions. In primary cortical neurons CIP4 is concentrated specifically at the tips of extending lamellipodia and filopodia, instead of endosomes as in other cell types. Overexpression of CIP4 results in lamellipodial protrusions around the cell body, subsequently delaying neurite formation and enlarging growth cones. These effects depend on the F-BAR and SH3 domains of CIP4 and on its ability to multimerize. Conversely, cortical neurons from CIP4-null mice initiate neurites twice as fast as controls. This is the first study to demonstrate that an F-BAR protein functions differently in neuronal vs. non-neuronal cells and induces lamellipodial protrusions instead of invaginations or filopodia-like structures.
Mitochondria are essential for neuronal survival and function. Proper degradation of aged and damaged mitochondria through mitophagy is a key cellular pathway for mitochondrial quality control. Recent studies have indicated that PINK1/Parkin-mediated pathways ensure mitochondrial integrity and function [1–8]. Translocation of Parkin to damaged mitochondria induces mitophagy in many non-neuronal cell types [9–16]. However, evidence showing Parkin translocation in primary neurons is controversial [9,15,17,18], leaving unanswered questions as to how and where Parkin-mediated mitophagy occurs in neurons. Here, we report the unique process of dissipating mitochondrial Δψm-induced and Parkin-mediated mitophagy in mature cortical neurons. Compared with non-neuronal cells, neuronal mitophagy is a much slower and compartmentally restricted process, coupled with reduced anterograde mitochondrial transport. Parkin-targeted mitochondria are accumulated in the somatodendritic regions where mature lysosomes are predominantly located. Time-lapse imaging shows dynamic formation and elimination of Parkin- and LC3-ring like structures surrounding depolarized mitochondria through the autophagy-lysosomal pathway in the soma. Knocking down Parkin in neurons impairs the elimination of dysfunctional mitochondria. Thus, our study provides neuronal evidence for dynamic and spatial Parkin-mediated mitophagy, which will help us understand whether altered mitophagy contributes to pathogenesis of several major neurodegenerative diseases characterized by mitochondrial dysfunction and impaired transport.
mitochondria; Parkin; lysosome; autophagosome; autophagy; depolarization; mitochondrial mobility; neuronal mitophagy; mitochondrial quality control
Humans and other species continually perform microscopic eye movements, even when attending to a single point [1-3]. These movements, which include microscopic drifts and microsaccades, are under control of the oculomotor system [2, 4, 5], elicit strong responses throughout the visual system [6-11], and have been thought to serve important functions [12-16]. The influence of these fixational eye movements on the acquisition and neural processing of visual information remains unknown. Here, we show that during viewing of natural scenes, microscopic eye movements carry out a crucial information-processing step: they remove predictable correlations in natural scenes by equalizing the spatial power of the retinal image within the frequency range of ganglion cells' peak sensitivity. This transformation, which had been attributed to center-surround receptive field organization [17-19], occurs prior to any neural processing, and reveals a form of matching between the statistics of natural images and those of normal eye movements. We further show that the combined effect of microscopic eye movements and retinal receptive field organization is to convert spatial luminance discontinuities into synchronous firing events, thus beginning the process of edge extraction. In sum, our results show that microscopic eye movements are fundamental to two goals of early visual processing —redundancy reduction [20, 21] and feature extraction— and, thus, that neural representations are intrinsically sensory-motor from the very first processing stages.
While humans have an incredible capacity to acquire new skills and alter their behavior as a result of experience, enhancements in performance are typically narrowly restricted to the parameters of the training environment, with little evidence of generalization to different, even seemingly highly related, tasks. Such specificity is a major obstacle for the development of many real-world training or rehabilitation paradigms, which necessarily seek to promote more general learning. In contrast to these typical findings, research over the past decade has shown that training on ‘action video games’ produces learning that transfers well beyond the training task. This has led to substantial interest among those interested in rehabilitation, for instance, after stroke or to treat amblyopia, or training for various precision-demanding jobs, for instance, endoscopic surgery or piloting unmanned aerial drones. Although the predominant focus of the field has been on outlining the breadth of possible action-game-related enhancements, recent work has concentrated on uncovering the mechanisms that underlie these changes, an important first step towards the goal of designing and using video games for more definite purposes. Game playing may not convey an immediate advantage on new tasks (increased performance from the very first trial), but rather the true effect of action video game playing may be to enhance the ability to learn new tasks. Such a mechanism may serve as a signature of training regimens that are likely to produce transfer of learning.
Receptor-induced Ca2+ oscillations provide ‘digitized’ signals that confer precise activation of downstream targets. New studies reveal that STIM proteins — sensors of endoplasmic reticulum Ca2+ levels — cyclically translocate during oscillations, transiently coupling to activate cell-surface Ca2+ entry channels, resulting in a spatially unique signal that selectively triggers immediate-early gene expression.
The small GTPase Arf6 has been shown to regulate the post-endocytic trafficking of a subset of membrane proteins, including β1 integrins, and inhibition of Arf6 function impairs both cell adhesion and motility . The activity of Arf GTPases is regulated by a large family of guanine nucleotide exchange factors (GEFs) . Arf-GEP100/BRAG2 is a GEF with reported specificity for Arf6 in vitro , but it is otherwise poorly characterized. Here we report that BRAG2 exists in two ubiquitously expressed isoforms, which we call BRAG2a and BRAG2b, both of which can activate Arf6 in vivo. Depletion of endogenous BRAG2 by siRNA leads to dramatic effects in the cell periphery; one such effect is an accumulation of β1 integrin on the cell surface and a corresponding enhancement of cell attachment and spreading on fibronectin-coated substrates. In contrast, depletion of Arf6 leads to intracellular accumulation of β1 integrin and reduced adhesion and spreading. These findings suggest that Arf6 regulates both endocytosis and recycling of β1 integrins and that BRAG2 functions selectively to activate Arf6 during integrin internalization.
Sex-ratio meiotic drive occurs when males produce a predominance of X-chromosome bearing sperm and an inordinate number of daughters. A driving X causes highly female-biased sex ratios and the risk of extinction. Polyandry can rescue a population from extinction.
Podosomes are cytoskeletal-based structures involved in extracellular matrix (ECM) remodeling and cellular motility. A new study now implicates podosomes in pore formation during myoblast fusion.
The DNA damage response (DDR) and DNA repair are critical for maintaining genomic stability and evading many human diseases [1, 2]. Recent findings indicate accumulation of SUN1, a nuclear envelope (NE) protein, is a significant pathogenic event in Emery-Dreifuss muscular dystrophy and Hutchinson-Gilford progeria syndrome, both caused by mutations in LMNA [3, 4]. However, roles of mammalian SUN proteins in mitotic cell division and genomic stability are unknown. Here we report that the inner NE proteins SUN1 and SUN2 may play a redundant role in DDR. Mouse embryonic fibroblasts from Sun1−/−Sun2−/− mice displayed premature proliferation arrest in S phase of cell cycle, increased apoptosis and DNA damage, and decreased perinuclear heterochromatin, indicating genome instability. Furthermore, activation of ATM and H2A.X, early events in DDR, were impaired in Sun1−/−Sun2−/− fibroblasts. A biochemical screen identified interactions between SUN1/2 and DNA-dependent protein kinase (DNAPK) complex that functions in DNA nonhomologous end joining repair and possibly in DDR [2, 5, 6]. Knockdown of DNAPK reduced ATM activation in NIH3T3 cells, consistent with a potential role of SUN1/2-DNAPK interaction during DDR. SUN1/2 could affect DDR by localizing certain nuclear factors to the NE or by mediating the communication between nuclear and cytoplasmic events.
KASH-SUN complex; Genomic instability; DNA repair; DNAPK; Ku70; Ku80; DDR; H2A.X; ATM; HGPS
Estimating depth from binocular disparity is extremely precise and the cue does not depend on statistical regularities in the environment. Thus, disparity is commonly regarded as the best visual cue for determining 3D layout. But depth from disparity is only precise near where one is looking; it is quite imprecise elsewhere [1-4]. To overcome this imprecision away from fixation, vision resorts to using other depth cues—e.g., linear perspective, familiar size, aerial perspective. But those cues depend on statistical regularities in the environment and are therefore not always reliable . Depth from defocus blur relies on fewer assumptions and has the same geometric constraints as disparity , but different physiological constraints [7-14]. Hence, blur could in principle fill in the parts of visual space where disparity is imprecise . We tested this possibility with a depth-discrimination experiment. We found that disparity was more precise near fixation and that blur was indeed more precise away from fixation. When both cues were available, observers relied on the more informative one. Blur appears to play an important, previously unrecognized [16,17] role in depth perception. Our findings lead to a new hypothesis about the evolution of slit-shaped pupils and have noteworthy implications for the design and implementation of stereo 3D viewing systems.
The primary cilium is a microtubule-based organelle that senses extracellular signals as a cellular antenna . Primary cilia are found on many types of cells in our body and play important roles in development and physiology. Defects of primary cilia cause a broad class of human genetic diseases called ciliopathies. To gain new insights into ciliary functions and better understand the molecular mechanisms underlying ciliopathies, it is of high importance to generate a catalog of primary cilia proteins. In this study, we isolated primary cilia from mouse kidney cells by using a calcium shock method and identified 195 candidate primary cilia proteins by MudPIT (multidimensional protein identification technology), protein correlation profiling, and subtractive proteomic analysis. Based on comparisons with other proteomic studies of cilia, around 75% of our candidate primary cilia proteins are shared components with motile or specialized sensory cilia. The remaining 25% of the candidate proteins are possible primary cilia specific proteins. These possible primary cilia specific proteins include Evc2, Inpp5e and Inversin, several of which have been linked to known ciliopathies. We have performed the first reported proteomic analysis of primary cilia from mammalian cells. These results provide new insights into primary cilia structure and function.
Many animals extract specific cues from rich visual scenes to guide appropriate behaviors. Such cues include visual motion signals produced both by self movement and by moving objects in the environment. The complexity of these signals requires neural circuits to link particular patterns of motion to specific behavioral responses.
Through electrophysiological recordings, we characterize genetically identified neurons in the optic lobe of Drosophila that are specifically tuned to detect motion signals produced by looming objects on a collision course with the fly. Using a genetic manipulation to specifically silence these neurons, we demonstrate that signals from these cells are important for flies to efficiently initiate the loom escape response. Moreover, through targeted expression of Channelrhodopsin in these cells, in flies that are blind, we reveal that optogenetic stimulation of these neurons is typically sufficient to elicit escape, even in the absence of any visual stimulus.
In this compact nervous system, a small group of neurons that extract a specific visual cue from local motion inputs serve to trigger the ethologically appropriate behavioral response.
Localized actomyosin contraction couples with actin polymerization and cell-matrix adhesion to regulate cell protrusions and retract trailing edges of migrating cells. While many cells migrate in collective groups during tissue morphogenesis, mechanisms that coordinate actomyosin dynamics in collective cell migration are poorly understood. Migration of Drosophila border cells, a genetically tractable model for collective cell migration, requires non-muscle myosin-II (Myo-II). How Myo-II specifically controls border cell migration and how Myo-II is itself regulated is largely unknown.
We show that Myo-II regulates two essential features of border cell migration: 1) initial detachment of the border cell cluster from the follicular epithelium and 2) the dynamics of cellular protrusions. We further demonstrate that the cell polarity protein Par-1 (MARK), a serine-threonine kinase, regulates the localization and activation of Myo-II in border cells. Par-1 binds to myosin phosphatase and phosphorylates it at a known inactivating site. Par-1 thus promotes phosphorylated Myosin Regulatory Light Chain (MRLC), thereby increasing Myo-II activity. Furthermore, Par-1 localizes to and increases active Myo-II at the cluster rear to promote detachment; in the absence of Par-1, spatially distinct active Myo-II is lost.
We identify a critical new role for Par-1 kinase: spatiotemporal regulation of Myo-II activity within the border cell cluster through localized inhibition of myosin phosphatase. Polarity proteins such as Par-1, which intrinsically localize, can thus directly modulate the actomyosin dynamics required for border cell detachment and migration. Such a link between polarity proteins and cytoskeletal dynamics may also occur in other collective cell migrations.
visual spatial frequency; auditory amplitude-modulation rate; auditory-visual interactions
Peripheral axons of somatosensory neurons innervate the skin early in development to detect touch stimuli. Embryological experiments had suggested that the skin produces guidance cues that attract sensory axons, but neither the attractants nor their neuronal receptors had previously been identified.
To investigate peripheral axon navigation to the skin, we combined live imaging of developing zebrafish Rohon-Beard (RB) neurons with molecular loss-of-function manipulations. Simultaneously knocking down two members of the LAR family of receptor tyrosine phosphatases expressed in RB neurons, or inhibiting their function with dominant negative proteins, misrouted peripheral axons to internal tissues. Time-lapse imaging indicated that peripheral axon guidance, rather than outgrowth or maintenance, was defective in LAR deficient neurons. Peripheral axons displayed a similar misrouting phenotype in mutants defective in heparan sulfate proteoglycan (HSPG) production and avoided regions in which HSPGs were locally degraded.
HSPGs and LAR family receptors are required for sensory axon guidance to the skin. Together, our results support a model in which peripheral HSPGs are attractive ligands for LAR receptors on RB neurons.
During spermatogenesis, germ cells initially expand exponentially through mitoses. A majority of these cells are then eliminated through p53-mediated apoptosis to maintain germline homeostasis [1–4]. However, the activity of p53 must be precisely modulated, especially suppressed in postmitotic spermatogenic cells, to guarantee robustness of spermatogenesis. Currently, how the suppression is achieved is not understood. Here, we show that Pumilio 1, a posttranscriptional regulator, binds to mRNAs representing 1527 genes, with significant enrichment for mRNAs involved in pathways regulating p53, cell cycle, and MAPK signaling. Particularly, eight mRNAs encoding activators of p53 are repressed by Pumilio 1. Deleting Pumilio 1 results in strong activation of p53 and apoptosis mostly in spermatocytes, which disrupts sperm production and fertility. Removing p53 reduces apoptosis and rescues testicular hypotrophy in Pumilio 1-null mice. These results indicate that key components of the p53 pathway are coordinately regulated by Pumilio 1 at the posttranscriptional level, which may exemplify an RNA operon.
Pumilio 1; RNA operon; spermatogenesis; apoptosis; p53; translational regulation
Cellular response to osmotic stress is critical for survival and involves volume control through the regulated transport of osmolytes [1–3]. Organelles may respond similarly to abrupt changes in cytoplasmic osmolarity [4–6]. The plastids of the Arabidopsis thaliana leaf epidermis provide a model system for the study of organellar response to osmotic stress within the context of the cell. An Arabidopsis mutant lacking two plastid-localized homologs of the bacteria mechanosensitive channel MscS (MscS-Like (MSL) 2 and 3) exhibits large round epidermal plastids that lack dynamic extensions known as stromules . This phenotype is present under normal growth conditions and does not require exposure to extracellular osmotic stress. Here, we show that increasing cytoplasmic osmolarity through a genetic lesion known to produce elevated levels of soluble sugars, exogenously providing osmolytes in the growth media, or withholding water rescues the msl2-1 msl3-1 leaf epidermal plastid phenotype, producing plastids that resemble the wild type in shape and size. Furthermore, the epidermal plastids in msl2-1 msl3-1 leaves undergo rapid and reversible volume and shape changes in response to extracellular hypertonic or hypotonic challenges. We conclude that plastids are under hypoosmotic stress during normal plant growth and dynamic response to this stress requires MSL2 and MSL3.
Exposure of human skin to solar ultraviolet radiation (UVR), a powerful carcinogen  comprising ~95% UVA and ~5% UVB at the Earth’s surface, promotes melanin synthesis in epidermal melanocytes [2, 3], which protects skin from DNA damage [4, 5]. UVB causes DNA lesions  that lead to transcriptional activation of melanin-producing enzymes, resulting in delayed skin pigmentation within days . In contrast, UVA causes primarily oxidative damage  and leads to immediate pigment darkening (IPD) within minutes, via an unknown mechanism [9, 10]. No receptor protein directly mediating phototransduction in skin has been identified. Here we demonstrate that exposure of primary human epidermal melanocytes (HEMs) to UVA causes calcium mobilization and early melanin synthesis. Calcium responses were abolished by treatment with G protein or PLC inhibitors, or by depletion of intracellular calcium stores. We show that the visual photopigment rhodopsin  is expressed in HEMs and contributes to UVR phototransduction. Upon UVR exposure, significant melanin production was measured within one hour; cellular melanin continued to increase in a retinal- and calcium-dependent manner up to five-fold after 24 hours. Our findings identify a novel UVA-sensitive signaling pathway in melanocytes that leads to calcium mobilization and melanin synthesis, and may underlie the mechanism of IPD in human skin.
HEN1-mediated 2′-O-methylation has been shown to be a key mechanism to protect plant microRNAs (miRNAs) and small interfering RNAs (siRNAs) as well as animal piwi-interacting RNAs (piRNAs) from degradation and 3′ terminal uridylation [1–8]. However, enzymes uridylating unmethylated miRNAs, siRNAs, or piRNAs in hen1 are unknown. In this study, a genetic screen identified a second-site mutation hen1 suppressor1-2 (heso1-2) that partially suppresses the morphological phenotypes of the hypomorphic hen1-2 allele and the null hen1-1 allele in Arabidopsis. HESO1 encodes a terminal nucleotidyl transferase that prefers to add untemplated uridine to the 3′ end of RNA, which is completely abolished by 2′-O-methylation. heso1-2 affects the profile of u-tailed miRNAs and siRNAs and increases the abundance of truncated and/or normal sized ones in hen1, which often results in increased total amount of miRNAs and siRNAs in hen1. In contrast, overexpressing HESO1 in hen1-2 causes more severe morphological defects and less accumulation of miRNAs. These results demonstrate that HESO1 is an enzyme uridylating unmethylated miRNAs and siRNAs in hen1. These observations also suggest that uridylation may destabilize unmethylated miRNAs through an unknown mechanism and compete with 3′-to-5′ exoribonuclease activities in hen1. This study shall have implications on piRNA uridylation in hen1 in animals.
Myosin IIIA (MYO3A) targets actin protrusion tips using a motility mechanism dependent on both motor and tail actin-binding activity . We show that myosin IIIB (MYO3B) lacks tail actin-binding activity and is unable to target COS7 cell filopodia tips, yet is somehow able to target stereocilia tips. Strikingly, when MYO3B is coexpressed with espin-1 (ESPN1), a MYO3A cargo protein endogenously expressed in stereocilia , MYO3B targets and carries ESPN1 to COS7 filopodia tips. We show that this tip-localization is lost when we remove the ESPN1 C-terminus actin-binding site. We also demonstrate that, like MYO3A , MYO3B can elongate filopodia by transporting ESPN1 to the polymerizing end of actin filaments. The mutual dependence of MYO3B and ESPN1 for tip-localization reveals a novel mechanism for the cell to regulate myosin tip-localization via a reciprocal relationship with cargo that directly participates in actin binding for motility. Our results are consistent with a novel form of motility for class III myosins that requires both motor and tail domain actin-binding activity, and show that the actin-binding tail can be replaced by actin-binding cargo. This study also provides a framework to better understand the late-onset hearing loss phenotype in patients with MYO3A mutations.
Visual perception involves information flow from lower to higher-order cortical areas, which are known to process different kinds of information. How does this functional specialization arise? As a step toward addressing this question, we combined fluorescent retrograde tracing with in vivo two-photon calcium imaging to simultaneously compare the tuning properties of neighboring neurons in areas 17 and 18 of ferret visual cortex that have different higher cortical projection targets.
Neurons projecting to the Posterior Suprasylvian Sulcus (PSS) were more direction-selective and preferred shorter stimuli, higher spatial frequencies, and higher temporal frequencies than neurons projecting to area 21, anticipating key differences between the functional properties of the target areas themselves. These differences could not be explained by a correspondence between anatomical and functional clustering within early visual cortex, and the largest differences were in properties generated within early visual cortex (direction selectivity and length preference) rather than in properties present in its retinogeniculate inputs.
These projection cell groups, and hence the higher-order visual areas to which they project, likely obtain their functional properties not from biased retinogeniculate inputs but from highly specific circuitry within the cortex.
Domestic pigeons are spectacularly diverse and exhibit variation in more traits than any other bird species . In The Origin of Species, Charles Darwin repeatedly calls attention to the striking variation among domestic pigeon breeds – generated by thousands of years of artificial selection on a single species by human breeders – as a model for the process of natural divergence among wild populations and species . Darwin proposed a morphology-based classification of domestic pigeon breeds , but the relationships among major groups of breeds and their geographic origins remain poorly understood [4, 5]. We used a large, geographically diverse sample of 361 individuals from 70 domestic pigeon breeds and two free-living populations to determine genetic relationships within this species. We found unexpected relationships among phenotypically divergent breeds that imply convergent evolution of derived traits in several breed groups. Our findings also illuminate the geographic origins of breed groups in India and the Middle East, and suggest that racing breeds have made substantial contributions to feral pigeon populations.
Epithelial tissues undergo extensive collective movements during morphogenesis, repair and renewal. Collective epithelial cell migration requires the intercellular coordination of cell-cell adhesions and the establishment of anterior-posterior polarity, while maintaining apical-basal polarity, but how this is achieved at the molecular level is not well understood.
Using an RNAi-based screen to identify Rho family GTPase regulators required for the collective migration of human bronchial epithelial cells, we identified myosin-IXA (gene name: Myo9a). Depletion of myosin-IXA, a RhoGAP and actin motor protein, in collectively migrating cells led to altered organization of the actin cytoskeleton and tension-dependent disruption of cell-cell adhesions, followed by an inability to form new adhesions resulting in cell scattering. Closer examination revealed myosin-IXA is required during the formation of junction-associated actin bundles soon after cell-cell contact. Structure-function analysis of myosin-IXA revealed that the motor domain is necessary and sufficient for binding to actin filaments, while expression of the RhoGAP domain partially rescued the cell scattering phenotype induced by myosin-IXA depletion. Finally, a FRET biosensor revealed a significant increase in Rho activity at nascent cell-cell contacts in myosin-IXA depleted cells compared to controls.
We propose that myosin-IXA locally regulates Rho and the assembly of thin actin bundles associated with nascent cell-cell adhesions and that this is required to sustain the collective migration of epithelial cells.