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The development of a fertilized egg to an embryo requires the proper temporal control of gene expression. During cell differentiation, timing is often controlled via cascades of transcription factors (TFs). However, in early development, transcription is often inactive, and many TF levels stay constant, suggesting that alternative mechanisms govern the observed rapid and ordered onset of gene expression. Here, we find that in early embryonic development access of maternally deposited nuclear proteins to the genome is temporally ordered via importin affinities, thereby timing the expression of downstream targets. We quantify changes in the nuclear proteome during early development and find that nuclear proteins, such as TFs and RNA polymerases, enter the nucleus sequentially. Moreover, we find that the timing of nuclear proteins’ access to the genome corresponds to the timing of downstream gene activation. We show that the affinity of proteins to importin is a major determinant in the timing of protein entry into embryonic nuclei. Thus, we propose a mechanism by which embryos encode the timing of gene expression in early development via biochemical affinities. This process could be critical for embryos to organize themselves before deploying the regulatory cascades that control cell identities. Here the authors address how embryos control the timing of specific gene activation in early frog development. They find transcription factors for early gene activation are maternally loaded and remain at constant levels, and rather that order of activation is based on their sequential entry into the nucleus based largely on their respective affinity to importins.

Exosomes are nanoscale vesicles that mediate intercellular communication. Cellular exosome uptake mechanisms are not well defined partly due to the lack of specific inhibitors of this complex cellular process. Exosome uptake depends on cholesterol-rich membrane microdomains called lipid rafts and can be blocked by non-specific depletion of plasma membrane cholesterol. Scavenger receptor type B-1 (SR-B1), found in lipid rafts, is a receptor for cholesterol-rich high-density lipoproteins (HDL). We hypothesized that a synthetic nanoparticle mimic of HDL (HDL NP) that binds SR-B1 and removes cholesterol through this receptor would inhibit cellular exosome uptake. In cell models, our data show that HDL NPs bind SR-B1, activate cholesterol efflux and attenuate the influx of esterified cholesterol. As a result, HDL NP treatment results in decreased dynamics and clustering of SR-B1 contained in lipid rafts and potently inhibits cellular exosome uptake. Thus, SR-B1 and targeted HDL NPs provide a fundamental advance in studying cholesterol-dependent cellular uptake mechanisms.

Self-organization of and by the cytoskeleton is central to the biology of the cell. Since their introduction in the early 1980s, cytoplasmic extracts derived from the eggs of the African clawed-frog, Xenopus laevis, have flourished as a major experimental system to study the various facets of cytoskeleton-dependent self-organization. Over the years, the many investigations that have used these extracts uniquely benefited from their simplified cell cycle, large experimental volumes, biochemical tractability and cell-free nature. Here, we review the contributions of egg extracts to our understanding of the cytoplasmic aspects of self-organization by the microtubule and the actomyosin cytoskeletons as well as the importance of cytoskeletal filaments in organizing nuclear structure and function.

Peptidylarginine deiminases (PADs or PADIs) catalyze the conversion of positively charged arginine to neutral citrulline, which alters target protein structure and function. Our previous work established that gonadotropin-releasing hormone agonist (GnRHa) stimulates PAD2-catalyzed histone citrullination to epigenetically regulate gonadotropin gene expression in the gonadotrope-derived LβT2 cell line. However, PADs are also found in the cytoplasm. Given this, we used mass spectrometry (MS) to identify additional non-histone proteins that are citrullinated following GnRHa stimulation and characterized the temporal dynamics of this modification. Our results show that actin and tubulin are citrullinated, which led us to hypothesize that GnRHa might induce their citrullination to modulate cytoskeletal dynamics and architecture. The data show that 10 nM GnRHa induces the citrullination of β-actin, with elevated levels occurring at 10 min. The level of β-actin citrullination is reduced in the presence of the pan-PAD inhibitor biphenyl-benzimidazole-Cl-amidine (BB-ClA), which also prevents GnRHa-induced actin reorganization in dispersed murine gonadotrope cells. GnRHa induces the citrullination of β-tubulin, with elevated levels occurring at 30 min, and this response is attenuated in the presence of PAD inhibition. To examine the functional consequence of β-tubulin citrullination, we utilized fluorescently tagged end binding protein 1 (EB1-GFP) to track the growing plus end of microtubules (MT) in real time in transfected LβT2 cells. Time-lapse confocal microscopy of EB1-GFP reveals that the MT average lifetime increases following 30 min of GnRHa treatment, but this increase is attenuated by PAD inhibition. Taken together, our data suggest that GnRHa-induced citrullination alters actin reorganization and MT lifetime in gonadotrope cells.

The dogma of coupled transcription and translation in bacteria has been challenged by recent reports of spatial segregation of these processes within the relatively simple cellular organization of the model organisms Escherichia coli and Bacillus subtilis . The bacterial species Gemmata obscuriglobus possesses an extensive endomembrane system. The membranes generate a very convoluted intracellular architecture in which some of the cell’s ribosomes appear to have less direct access to the cell’s nucleoid(s) than others. This observation prompted us to test the hypothesis that a substantial proportion of G. obscuriglobus translation may be spatially segregated from transcription. Using immunofluorescence and immunoelectron microscopy, we showed that translating ribosomes are localized throughout the cell, with a quantitatively greater proportion found in regions distal to nucleoid(s). Our results extend information about the phylogenetic and morphological diversity of bacteria in which the spatial organization of transcription and translation has been studied. These findings also suggest that endomembranes may provide an obstacle to colocated transcription and translation, a role for endomembranes that has not been reported previously for a prokaryotic organism. Our studies of G. obscuriglobus may provide a useful background for consideration of the evolutionary development of eukaryotic cellular complexity and how it led to decoupled processes of gene expression in eukaryotes.

The role of neuropeptides in nervous system function is still in many cases undefined. In the present study we examined a possible role of the 36–amino acid neuropeptide Y (NPY) with regard to three functions: axon guidance and attraction/repulsion, adult neurogenesis, and control of food intake. Growth cones from embryonic dorsal root ganglion neurons were studied in culture during asymmetrical gradient application of NPY. Growth cones were monitored over a 60-min period, and final turning angle and growth rate were recorded. In the second part the NPY Y1 and Y2 receptors were studied in the subventricular zone, the rostral migratory stream, and the olfactory bulb in normal mice and mice with genetically deleted NPY Y1 or Y2 receptors. In the third part an anorectic mouse was analyzed with immunohistochemistry. 1) NPY elicited an attractive turning response and an increase in growth rate, effects exerted via the NPY Y1 receptor. 2) The NPY Y1 receptor was expressed in neuroblasts in the anterior rostral migratory stream. Mice deficient in the Y1 or Y2 receptor had fewer proliferating precursor cells and neuroblasts in the subventricular zone and rostral migratory stream and fewer neurons in the olfactory bulb expressing calbindin, calretinin or tyrosine hydroxylase. 3) In the anorectic mouse markers for microglia were strongly upregulated in the arcuate nucleus and in projection areas of the NPY/agouti gene-related protein arcuate system. NPY participates in several mechanisms involved in the development of the nervous system and is of importance in the control of food intake.

The development of a fertilized egg to an embryo requires the proper temporal control of gene expression. During cell differentiation, timing is often controlled via cascades of transcription factors (TFs). However, in early development, transcription is often inactive, and many TF levels are constant, suggesting that unknown mechanisms govern the observed rapid and ordered onset of gene expression. Here, we find that in early embryonic development, access of maternally deposited nuclear proteins to the genome is temporally ordered via importin affinities, thereby timing the expression of downstream targets. We quantify changes in the nuclear proteome during early development and find that nuclear proteins, such as TFs and RNA polymerases, enter nuclei sequentially. Moreover, we find that the timing of the access of nuclear proteins to the genome corresponds to the timing of downstream gene activation. We show that the affinity of proteins to importin is a major determinant in the timing of protein entry into embryonic nuclei. Thus, we propose a mechanism by which embryos encode the timing of gene expression in early development via biochemical affinities. This process could be critical for embryos to organize themselves before deploying the regulatory cascades that control cell identities. Competing Interest Statement The authors have declared no competing interest.

The microtubule (MT) cytoskeleton plays critically important roles in numerous cellular functions in eukaryotes, and it does so across a functionally diverse and morphologically disparate range of cell types. In these roles, MT assemblies must adopt distinct morphologies and physical dimensions to perform specific functions. As such, these macromolecular assemblies-as well as the dynamics of the individual MT polymers from which they are made-must scale and change in accordance with cell size and geometry. As first shown by Inoue using polarization microscopy, microtubules assemble to a steady state in mass, leaving enough of their subunits soluble to allow rapid growth and turnover. This suggests some negative feedback that limits the extent of assembly, for example decrease in growth rate, or increase in catastrophe rate, as the soluble subunit pool decreases. Such feedbacks might be global or local. Although these ideas have informed the field for decades, they have not been observed experimentally. Here we describe an experimental system designed to examine these long-standing ideas and determine a role for MT plus-end density in regulating MT growth rates.

The nerve growth cone is the motile tip of the nascent neurite. It navigates through the extracellular environment to predetermined targets. This process, called pathfinding, necessitates the growth cone's ability to respond to extracellular cues (both repulsive and attractive) that direct its orientation and advancement. Growth cone motility, like all amoeboid processes, is intrinsically dependent upon the temporal and spatial regulation of adhesion. However, little is known about the signaling cascades that ultimately control growth cone adhesion sites. Repellent cues are thought to evoke growth cone turning responses by causing localized, asymmetric “collapse” characterized by detachment from the substratum and rapid loss of growth cone area. The repellent Semaphorin3A (Sema3A) was used to induce either growth cone collapse or turning. Sema3A-induced collapse was found to require generation of the eicosanoid 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] and 12(S)-HETE alone was sufficient to induce a collapse response. Additionally, inhibition of protein kinase C (PKC), which is activated by 12(S)-HETE, resulted in growth cones refractory to Sema3A-induced turning. Furthermore, phosphorylation and translocation of the major PKC substrate in the brain, the myristoylated, alanine rich, C kinase substrate (MARCKS), was stimulated by 12(S)-HETE treatment in isolated growth cones. MARCKS co-localizes with integrins and tetraspanins at the growth cone periphery. Interestingly, growth cones expressing a phosphorylation- and translocation-deficient mutant MARCKS were not only refractory to 12(S)-HETE-induced collapse but responded to Sema3A by attraction rather than repulsion. These studies advance our understanding of the mechanisms by which adhesion is regulated during growth cone pathfinding. They define and characterize a novel, eicosanoid-mediated signaling cascade that targets growth cone adhesions. This work is the first (i) to establish a requirement for eicosanoid generation in repellent-mediated collapse, (ii) to demonstrate that PKC is a downstream effector in Sema3A repellent signaling, and (iii) to show that phosphorylation and translocation of the PKC substrate MARCKS are necessary for normal Sema3A-induced repulsion. These findings provide a causal link between PKC activation and growth cone turning and collapse. Lastly, this work defines a new type of growth cone adhesion containing integrins and tetraspanins that appears to be targeted by the eicosanoid-mediated pathway.

This paper describes an investigation into part of the mechanical mechanisms underlying the formation of mitotic spindle, the cellular machinery responsible for chromosomal separation during cell division. In normal eukaryotic cells, spindles are composed of microtubule filaments that radiate outward from two centrosomes. In many transformed cells, however, centrosome number is misregulated resulting in cells with more than two centrosomes. Addressing the question of how these cells accommodate these additional structures by coalescing supernumerary centrosomes to form normal spindles will provide a powerful insight toward understanding the proliferation of cancer cells and developing new therapeutics. The process of centrosome coalescence is thought to involve motor proteins that function to slide microtubules relative to one another. Here we use in vitro motility assays combined with fluorescence microscopy to visualize, characterize and quantify microtubule-microtubule interactions. After segmenting the microtubules, their speed and direction of movement are the extracted features to cluster their interaction type. In order to evaluate the potential of our processing algorithm, we created a simulated dataset similar to the time-lapse series. Once our procedure has been optimized using the simulated data, we will apply it to the real data. Results of our analyses will provide a quantitative description of interaction among microtubules. This is a potentially important step toward more thorough understanding of cancer.