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bioRxiv2017

A broad role for YBX1 in defining the small non-coding RNA composition of exosomes

RNA is secreted from cells enclosed within extracellular vesicles (EVs). Defining the RNA composition of EVs is challenging due to their co-isolation with contaminants, a lack of knowledge of the mechanisms of RNA sorting into EVs and limitations of conventional RNA-seq methods. Here we present our observations using thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) to characterize the RNA extracted from HEK293T cell EVs isolated by flotation gradient ultracentrifugation and from exosomes containing the tetraspannin CD63 further purified from the gradient fractions by immunoisolation. We found that EV-associated transcripts are dominated by full-length, mature tRNAs and other small non-coding RNAs encapsulated within vesicles. A substantial proportion of the reads mapping to protein-coding genes, long non-coding, and antisense RNAs were due to DNA contamination on the surface of vesicles. Nevertheless, sequences mapping to spliced mRNAs were identified within HEK293T cell EVs and exosomes, among the most abundant being transcripts containing a 5 terminal oligopyrimidine (5 TOP) motif. Our results indicate that the RNA-binding protein YBX1, which we showed previously is required for the sorting of selected miRNAs into exosomes, plays a role in the sorting of highly abundant small non-coding RNA species, including tRNAs, Y RNAs, and Vault RNAs. Finally, we obtained evidence for an EV-specific tRNA modification, perhaps indicating a role for post-transcriptional modification in the sorting of some RNA species into EVs. The identification of full-length small non-coding RNAs within EVs suggests a role for EVs in the export and possible intercellular functional transfer of abundant cellular transcripts.\n\nStatement of SignificanceCells release vesicles containing selectively packaged cargo, including RNA, into the extracellular environment. Prior studies have identified RNA inside extracellular vesicles (EVs) but, due to limitations of conventional sequencing methods, highly structured and post-transcriptionally modified RNA species were not effectively captured. Using an alternative sequencing approach (TGIRT-seq), we found that EVs contain abundant small non-coding RNA species, including full-length tRNAs and Y RNAs. Using a knockout cell line, we obtained evidence that the RNA-binding protein YBX1 plays a role in sorting small non-coding RNAs into a subpopulation of extracellular vesicles termed exosomes. These experiments expand our understanding of EV-RNA composition and provide insights into how RNA is sorted into EVs for export from the cell.

cell biology↗

bioRxiv2017

FAM192A/PIP30 regulates Cajal body dynamics by antagonizing PA28γ/coilin interaction

PA28{gamma} is a nuclear activator of the 20S proteasome involved in the regulation of several essential cellular processes, such as cell proliferation, apoptosis, nuclear dynamics and cellular stress response. Unlike the 19S regulator of the proteasome, which specifically recognizes ubiquitylated proteins, PA28{gamma} promotes the degradation of several substrates by the proteasome in an ATP- and ubiquitin-independent manner. However its exact mechanisms of action are unclear and likely to involve additional partners that remain to be identified. Here we report the identification of the first cofactor of PA28{gamma}, PIP30/FAM192A. PIP30 binds directly and specifically via its C-terminal end and in an interaction stabilized by casein kinase 2 phosphorylation to both free and 20S proteasome-associated PA28{gamma}. Its recruitment to proteasome-containing complexes depends on PA28{gamma} and its expression increases the association of PA28{gamma} with the 20S proteasome in cells. Further dissection of its possible roles shows that PIP30 alters PA28{gamma}-dependent activation of peptide degradation by the 20S proteasome in vitro and negatively controls in cells the presence of PA28{gamma} in Cajal Bodies by inhibition of its association with the key Cajal body component coilin. Altogether, our data show that PIP30 deeply affects PA28{gamma} interactions with cellular proteins, including 20S proteasome, demonstrating that it is an important regulator of PA28{gamma} in cells and thus a new player in the control of the multiple functions of the proteasome within the nucleus.\n\nSignificance StatementThe 20S proteasome is a key actor of the control of protein levels and integrity in cells. To perform its multiple functions, it works with a series of regulators, among which a nuclear complex called PA28{gamma}. In particular, PA28{gamma} participates in the regulation of cell proliferation and nuclear dynamics. We describe here the characterization of a novel protein, PIP30/FAM192A, which binds tightly to PA28{gamma} and favors its interaction with the 20S proteasome while inhibiting its association with coilin, a central component of nuclear Cajal bodies. Thus PIP30/FAM192A critically controls the interactome and consequently the functions of PA28{gamma}, and appears to be a new player in the fine regulation of intracellular proteostasis in the cell nucleus.

cell biology↗

bioRxiv2017

Drosophila PLP forms centriolar-clouds that promote centriole stability, cohesion and MT nucleation

Pericentrin is a conserved centrosomal protein whose dysfunction has been linked to several human diseases. The precise function of Pericentrin, however, is controversial. Here, we examine Drosophila Pericentrin-like- protein (PLP) function in vivo, in tissues that form both centrosomes and cilia. PLP mutant centrioles exhibit four major defects: (1) They are too short and have subtle structural defects; (2) They separate prematurely, and so overduplicate; (3) They organise fewer MTs during interphase; (4) They fail to establish and/or maintain a proper connection to the plasma membrane-- although, surprisingly, mutant centrioles can still form an axoneme and recruit transition zone (TZ) proteins. We show that PLP helps to form \" pericentriolar clouds\" of electron-dense material that emanate from the central cartwheel spokes and spread outward to surround the mother centriole. The partial loss of these structures may explain the complex centriole, centrosome and cilium defects we observe in PLP mutant cells.

cell biology↗

bioRxiv2017

Langevin Dynamics Simulation of AKAP-PKA Complex: Re-Envisioning the Local Concentration Mechanism for Directing PKA Phosphorylation

The second messenger cAMP and its effector cAMP-dependent protein kinase A (PKA) constitute a ubiquitous cell signaling system. In its inactive state PKA is composed of two regulatory subunits that dimerize, and two catalytic subunits that are inhibited by the regulatory subunits. Activation of the catalytic subunits occurs upon binding of two molecules of cAMP to each regulatory subunit. Although many receptor types existing within the same cell may use this signaling system, compartmentation of signaling is thought to occur due to A-Kinase Anchoring Proteins (AKAPs), which act to co-localize PKA with specific substrates. However, the molecular mechanism allowing AKAPs to direct PKA phosphorylation to a particular substrate remained elusive, as prior evidence suggested that the catalytic subunit, which is highly diffusible, is released after cAMP binding to the regulatory subunit. Recent evidence from Smith et al. suggests that in the cell, the catalytic subunit may in fact not be released from the AKAP complex [1, 2]. They further demonstrated that alterations in the structure of the PKA regulatory subunit tether affect substrate phosphorylation. We use a novel computational software based on Langevin dynamics, SpringSaLaD, to simulate the AKAP-PKA complex in order to determine a molecular mechanism for the changes in phosphorylation seen with alteration in tether length and flexibility, and to demonstrate whether or not AKAPs can effectively direct PKA phosphorylation to a particular substrate upon release of the catalytic subunit from the complex. We find that short and flexible tethers contribute to a decrease in the average characteristic time of binding, allowing the catalytic subunit to spend more time in a bound state with the substrate, which yields faster characteristic times of phosphorylation. We further demonstrate that release of the catalytic subunit from the AKAP complex abrogates the effect of tethering, with characteristic times of phosphorylation similar to non-AKAP bound PKA. The data demonstrates that AKAPs likely do not release the catalytic subunit in directing PKA phosphorylation to AKAP bound substrates. In combination with the changes in characteristic time of phosphorylation which are driven by tether structure, this work indicates that the purpose of AKAPs may be to increase the efficiency of phosphorylation of particular AKAP substrates.

cell biology↗

bioRxiv2017

Curvature regulates subcellular organelle location to control intracellular signal propagation

The shape of the cell is connected to its function; however, we do not fully understand underlying mechanisms by which global shape regulates a cells functional capabilities. Using theory, experiments and simulation, we investigated how physiologically relevant cell shape changes affect subcellular organization, and consequently intracellular signaling, to control information flow needed for phenotypic function. Vascular smooth muscle cells going from a proliferative and motile circular shape to a contractile fusiform shape show changes in the location of the sarcoplasmic reticulum, inter-organelle distances and differential distribution of receptors in the plasma membrane. These factors together lead to the modulation of signals transduced by the M3 muscarinic receptor/Gq/PLC{beta} pathway at the plasma membrane, amplifying Ca2+ dynamics in the cytoplasm and the nucleus resulting in phenotypic changes, as determined by increased activity of myosin light chain kinase in the cytoplasm and enhanced nuclear localization of the transcription factor NFAT. Taken together, our observations show a systems level phenomenon whereby global cell shape affects subcellular organization to modulate signaling that enables phenotypic changes.

cell biology↗

bioRxiv2017

Sympathetic Transmitters Controlling Thermogenic Efficacy of Brown Adipocytes by Modulating Mitochondrial Complex V

Obesity is epidemic worldwide as the results of excessive energy intake or inefficient energy expenditure. It is promising to utilize the thermogenic function of brown adipose tissue for obesity intervention. However, the mechanisms controlling the efficacy of norepinephrine-induced thermogenesis in brown adipocytes remain elusive. Here, we demonstrate that the proton ATPase activity of mitochondrial complex V is a key factor, which antagonizes proton leak by UCP1 and determines the efficacy of norepinephrine-induced thermogenesis in brown adipocytes. Furthermore, to avoid unnecessary and undesired heat production, we reveal that ATP as a sympathetic cotransmitter is necessary for the high efficacy and specificity of norepinephrine-induced thermogenesis in brown adipocytes by upregulating the ATP synthase activity of complex V. Thus, we demonstrate the modulation mechanism of thermogenic efficacy in brown adipocytes. These findings imply new strategies for efficiently utilizing brown adipocytes thermogenic capacity, and therapeutic targets for the treatments of obesity and diabetes.\n\nHighlights1. Norepinephrine (NE) induces heterogeneous responses in brown adipocytes\n\n2. NE activates the H+-ATPase activity of mitochondrial complex V\n\n3. Mitochondrial complex V is a key factor in NE-induced thermogenic efficacy\n\n4. ATP as a sympathetic cotransmitter enhances the NE-induced thermogenic efficacy

cell biology↗

bioRxiv2017

Flat-to-curved transition during clathrin-mediated endocytosis correlates with a change in clathrin-adaptor ratio and is regulated by membrane tension

Although essential for many cellular processes, the sequence of structural and molecular events during clathrin-mediated endocytosis remains elusive. While it was believed that clathrin-coated pits grow with a constant curvature, it was recently suggested that clathrin first assembles to form a flat structure and then bends while maintaining a constant surface area. Here, we combine correlative electron and light microscopy and mathematical modelling to quantify the sequence of ultrastructural rearrangements of the clathrin coat during endocytosis in mammalian cells. We confirm that clathrin-coated structures can initially grow flat and that lattice curvature does not show a direct correlation with clathrin coat assembly. We demonstrate that curvature begins when 70% of the final clathrin content is acquired. We find that this transition is marked by a change in the clathrin to clathrin-adaptor protein AP2 ratio and that membrane tension suppresses this transition. Our results support the model that mammalian cells dynamically regulate the flat-to-curved transition in clathrin-mediated endocytosis by both biochemical and mechanical factors.

cell biology↗

bioRxiv2017

CRISPR/Cas9-mediated Knock-in of an Optimized TetO Repeat for Live Cell Imaging of Endogenous Loci

Nuclear organization has an important role in determining genome function; however, it is not clear how spatiotemporal organization of the genome relates to functionality. To elucidate this relationship, a high-throughput method for tracking any locus of interest is desirable. Here, we report an efficient and scalable method named SHACKTeR (Short Homology and CRISPR/Cas9-mediated Knock-in of a TetO Repeat) for live cell imaging of specific chromosomal regions. Compared to alternatives, our method does not require a nearby repetitive sequence and it requires only two modifications to the genome: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat and its visualization by TetR-EGFP expression. Our simplified knock-in protocol, utilizing short homology arms integrated by PCR, was successful at labeling 9 different loci in HCT116 cells with up to 20% efficiency. These loci included both nuclear speckle-associated, euchromatin regions and nuclear lamina-associated, heterochromatin regions. We anticipate the general applicability and scalability of our method will enhance causative analyses between gene function and compartmentalization in a high-throughput manner.

cell biology↗

bioRxiv2017

Rapid isolation of functionally intact nuclei from the yeast Saccharomyces

Most available methods for nuclear isolation entail lengthy procedures that are difficult to master and generally emphasize yield and enrichment over nuclear preservation, thus limiting their utility for further studies. Here we demonstrate a novel and robust method to rapidly isolate well-preserved yeast nuclei. The method can be easily adapted to multiple preparation scales depending on experimental need and it can readily be performed on multiple samples by a single researcher in one day. We show that the nuclei fraction is strongly enriched and that the resulting nuclei are free from contaminating endoplasmatic reticulum and other cell debris. EM studies show that preservation of nuclear morphology is exquisite, making it possible to study peripheral nuclear pore components such as the cytoplasmic filaments and the basket, whose structure is generally difficult to maintain ex vivo. In addition, incubation of isolated nuclei with bulk transport substrates of different sizes and with import cargo indicates that the nuclear envelope is intact and nuclear pores retain their capacity to bind transport substrates. Our results suggest that this preparation procedure will greatly facilitate studies of the yeast nucleus which have been difficult to establish and to multiplex to date.

cell biology↗

bioRxiv2017

cAMP Signaling Regulates DNA Demethylation by Augmenting the Intracellular Labile Ferrous Iron Pool

It is widely accepted that cAMP regulates gene transcription principally by activating the protein kinase A (PKA)-targeted transcription factors. Here, we show that cAMP enhances the generation of 5-hydroxymethylcytosine (5hmC) in multiple cell types. 5hmC is converted from 5-methylcytosine (5mC) by Tet methylcytosine dioxygenases, for which Fe(II) is an essential cofactor. The promotion of 5hmC was mediated by a prompt increase of the intracellular labile Fe(II) pool (LIP). cAMP enhanced the acidification of endosomes for Fe(II) release to the LIP likely through RapGEF2. The effect of cAMP on Fe(II) and 5hmC was confirmed by adenylate cyclase activators, phosphodiesterase inhibitors, and most notably by stimulation of G protein-coupled receptors (GPCR). The transcriptomic changes caused by cAMP occurred in concert with 5hmC elevation in differentially transcribed genes. Collectively, these data show a previously unrecognized regulation of gene transcription by GPCR-cAMP signaling through augmentation of the intracellular labile Fe(II) pool and DNA demethylation.

cell biology↗

bioRxiv2017

Soluble Syntaxin 3 Functions as a Transcription Regulator

Syntaxins - a conserved family of SNARE proteins - contain C-terminal transmembrane anchors required for their membrane fusion activity. Here we show that syntaxin 3 (Stx3) unexpectedly also functions as a nuclear regulator of gene expression. Alternative splicing leads to a soluble isoform, termed Stx3S, lacking the transmembrane anchor. Soluble Stx3S binds to the nuclear import factor RanBP5, targets to the nucleus and interacts physically and functionally with several transcription factors, including ETV4 and ATF2. Stx3S is differentially expressed in normal human tissues, during epithelial cell polarization, and in breast cancer vs. normal breast tissue. Inhibition of endogenous Stx3S expression leads to changes in the expression of cancer-associated genes and promotes cell proliferation. Similar nuclear-targeted, soluble forms of other syntaxins were identified suggesting that nuclear signaling is a conserved, novel function common among these membrane trafficking proteins.

cell biology↗

bioRxiv2017

Specification of the germline by Nanos-dependent down-regulation of the somatic synMuvB transcription factor LIN-15B

The Nanos RNA-binding protein has been implicated in the specification of primordial germ cells (PGCs) in metazoans, but the underlying mechanisms remain poorly understood. We have profiled the transcriptome of PGCs lacking the nanos homologues nos-1 and nos-2 iC. elegans. nos-1nos-2 PGCs fail to silence hundreds of genes normally expressed in oocytes and somatic cells, a phenotype reminiscent of PGCs lacking the repressive PRC2 complex. The nos-1nos-2 phenotype depends on LIN-15B, a broadly expressed synMuvB class transcription factor known to antagonize PRC2 activity in somatic cells. LIN-15B is maternally-inherited by all embryonic cells and is down-regulated specifically in PGCs in a nos-1nos-2-dependent manner. Consistent with LIN-15B being a critical target of Nanos regulation, inactivation of maternal LIN-15B restores fertility to nos-1nos-2 mutants. These studies demonstrate a central role for Nanos in reprogramming the transcriptome of PGCs away from an oocyte/somatic fate by down-regulating an antagonist of PRC2 activity.

cell biology↗

bioRxiv2017

Cell-nonautonomously tunable actomyosin flowsorient distinct cell division axes

Cell division axes during animal development are arranged in diverse orientations, but the molecular mechanisms underlying this diversity remain unclear. By focusing on oriented divisions that are independent of known microtubule/dynein pathways, we show here that the non-muscle myosin II motor is an extrinsically tunable force generator that orients cell division axes through cortical actomyosin flows. We identified three extracellular cues that generate different actomyosin flows. A single contact site locally inhibited myosin activity in a mechanosensitive manner to generate local flow asymmetry, while size asymmetry of two contact sites and Wnt signaling both polarized myosin activity and actomyosin flow, with the latter overriding mechanosensitive effects. These intracellular actomyosin flow anisotropies specify distinct division axes to establish the geometries of not only Caenorhabditis elegans 4-, 6-, and 7-cell stage but also mouse 4-cell stage embryos. Tunable actomyosin flows together with microtubule/dynein pathways may specify diverse division axes across species.

cell biology↗

bioRxiv2017

The activation loop residue serine 173 of S.pombe Chk1 kinase is critical for the response to DNA replication stress

Why the DNA damage checkpoint kinase Chk1 protects the genome of lower and higher eukaryotic cells differentially is still unclear. Mammalian Chk1 regulates replication origins, safeguards DNA replication forks and promotes fork progression. Conversely, yeast Chk1 acts only in G1 and G2. We report here that the mutation of serine 173 (S173A) in the activation loop of fission yeast Chk1 abolishes the G1-M and S-M checkpoints without affecting the G2-M arrest. Although Chk1-S173A is fully phosphorylated at serine 345 by the DNA damage sensor Rad3 (ATR) when DNA replication forks break, cells fail to stop the cell cycle. Mutant cells are uniquely sensitive to the DNA alkylation agent methyl- methanesulfate (MMS). This MMS sensitivity is genetically linked with the lagging strand DNA polymerase delta. Chk1-S173A is also unable to block mitosis when the G1 transcription factor Cdc10 is impaired. Serine 173 is equivalent to lysine 166 in human Chk1, an amino acid important for substrate specificity. We conclude that the removal of serine 173 impairs the phosphorylation of a Chk1 target that is important to protect cells from DNA replication stress.\n\nSummary statementMutation of serine-173 in the activation loop of Chk1 kinase may promote cancer as it abolishes the response to genetic alterations that arise while chromosomes are being copied.

cell biology↗

bioRxiv2017

Rapid DNA Replication Origin Licensing Protects Stem Cell Pluripotency

Complete and robust human genome duplication requires loading MCM helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single cell analyses we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation towards all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.

cell biology↗

bioRxiv2017

Compensation For Chronic Oxidative Stress In ALADIN Null Mice

BackgroundMutations in the AAAS gene coding for the nuclear pore complex protein ALADIN lead to the autosomal recessive disorder triple A syndrome. Triple A patients present with a characteristic phenotype including alacrima, achalasia and adrenal insufficiency. Patient fibroblasts show increased levels of oxidative stress and several in vitro studies demonstrated that the nucleoporin ALADIN is involved in the cellular oxidative stress response and in adrenal steroidogenesis. We showed that ALADIN knock-out mice lack a phenotype resembling human triple A syndrome. Thus, we hypothesized that application of chronic oxidative stress by ingestion of paraquat will generate triple A-like phenotype in ALADIN null mice.\n\nResultsWe demonstrate that ALADIN knock-out mice present with an unexpected compensated glutathione metabolism still lacking a phenotype resembling human triple A syndrome after application of chronic oxidative stress. We could not observe increased levels of oxidative stress and alterations in adrenal steroidogenesis in mice depleted for ALADIN.\n\nConclusionsThis study stresses the species-specific role of the nucleoporin ALADIN presenting a novel compensatory mechanism of the cellular glutathione redox response and shedding light on the role of ALADIN in the cell.

cell biology↗

bioRxiv2017

CYRI (FAM49) proteins are local inhibitors of Scar/WAVE induced lamellipodia that bind directly to active Rac1

Actin-based protrusions driving cell migration are reinforced through positive feedback, but it is unclear how the cell restricts the eventual size of a protrusion or limits positive signals to cause splitting or retraction. We have identified an evolutionarily conserved regulator of the protrusion machinery, which we name CYRI (CYFIP-related Rac interacting) protein. CYRI shows sequence similarity to the Scar/WAVE complex member CYFIP in a Domain of Unknown Function, DUF1394. CYRI binds specifically to activated Rac1 via a common motif shared with CYFIP, establishing DUF1394 as a new Rac1 binding domain. CYRI-depleted cells have broad, Scar/WAVE-enriched lamellipodia and enhanced Rac1 signaling. Conversely, CYRI overexpression suppresses spreading and dramatically sharpens protrusions into unproductive needles. CYRI proteins use dynamic inhibition of Scar/WAVE induced actin to focus positive protrusion signals and regulate pseudopod complexity. CYRI behaves like a \"local inhibitor\" predicted and described in widely accepted mathematical models, but not previously identified in living cells.

cell biology↗

bioRxiv2017

Mono-ubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes

Syntaxin 3 (Stx3), a SNARE protein located and functioning at the apical plasma membrane of epithelial cells, is required for epithelial polarity. A fraction of Stx3 is localized to late endosomes / lysosomes though how it traffics there and its function in these organelles is unknown. Here we report that Stx3 undergoes mono - ubiquitination in a conserved polybasic domain. Stx3 present at the basolateral - but not the apical - plasma membrane is rapidly endocytosed, targeted to endosomes, internalized into intraluminal vesicles (ILVs) and excreted in exosomes. A non - ubiquitinatable mutant of Stx3 (Stx3 - 5R) fails to enter this pathway and leads to the inability of the apical exosomal cargo protein GPRC5B to enter the ILV / exosomal pathway. This suggests that ubiquitination of Stx3 leads to removal from the basolateral membrane to achieve apical polarity, that Stx3 plays a role in the recruitment of cargo to exosomes, and that the Stx3 - 5R mutant acts as a dominant - negative inhibitor. Human cytomegalovirus (HCMV) acquires its membrane in an intracellular compartment and we show that Stx3 - 5R strongly reduces the number of excreted infectious viral particles. Altogether these results suggest that Stx3 functions in the transport of specific proteins to apical exosomes and that HCMV exploit this pathway for virion excretion.

cell biology↗