A novel strategy to uncover specific GO terms/phosphorylation pathways in phosphoproteomic data in Arabidopsis thaliana

Background Proteins are the workforce of the cell and their phosphorylation status tailors specific responses efficiently. One of the main challenges of phosphoproteomic approaches is to deconvolute biological processes that specifically respond to an experimental query from a list of phosphoproteins. Comparison of the frequency distribution of GO (Gene Ontology) terms in a given phosphoproteome set with that observed in the genome reference set (GenRS) is the most widely used tool to infer biological significance. Yet, this comparison assumes that GO term distribution between the phosphoproteome and the genome are identical. However, this hypothesis has not been tested due to the lack of a comprehensive phosphoproteome database. Results In this study, we test this hypothesis by constructing three phosphoproteome databases in Arabidopsis thaliana: one based in experimental data (ExpRS), another based in in silico phosphorylation protein prediction (PredRS) and a third that is the union of both (UnRS). Our results show that the three phosphoproteome reference sets show default enrichment of several GO terms compared to GenRS, indicating that GO term distribution in the phosphoproteomes does not match that of the genome. Moreover, these differences overshadow the identification of GO terms that are specifically enriched in a particular condition. To overcome this limitation, we present an additional comparison of the sample of interest with UnRS to uncover GO terms specifically enriched in a particular phosphoproteome experiment. Using this strategy, we found that mRNA splicing and cytoplasmic microtubule compounds are important processes specifically enriched in the phosphoproteome of dark-grown Arabidopsis seedlings. Conclusions This study provides a novel strategy to uncover GO specific terms in phosphoproteome data of Arabidopsis that could be applied to any other organism. We also highlight the importance of specific phosphorylation pathways that take place during dark-grown Arabidopsis development.

The short flagella 1 (SHF1) gene in Chlamydomonas encodes a Crescerin TOG-domain protein required for late stages of flagellar growth

Length control of flagella represents a simple and tractable system to investigate the dynamics of organelle size. Models for flagellar length control in the model organism, Chlamydomonas reinhardtii have focused on the length-dependence of the intraflagellar transport (IFT) system which manages the delivery and removal of axonemal subunits at the tip of the flagella. One of these cargoes, tubulin, is the major axonemal subunit, and its frequency of arrival at the tip plays a central role in size control models. However, the mechanisms determining tubulin dynamics at the tip are still poorly understood. We discovered a loss-of-function mutation that leads to shortened flagella, and found that this was an allele of a previously described gene, SHF1, whose molecular identity had not previously been determined. We found that SHF1 encodes a Chlamydomonas ortholog of Crescerin, previously identified as a cilia-specific TOG-domain array protein that can bind tubulin via its TOG domains and increase tubulin polymerization rates. In this mutant, flagellar regeneration occurs with the same initial kinetics as wild-type cells, but plateaus at a shorter length. Using a computational model in which the flagellar microtubules are represented by a differential equation for flagellar length combined with a stochastic model for cytoplasmic microtubule dynamics, we found that our experimental results are best described by a model in which Crescerin/SHF1 binds tubulin dimers in the cytoplasm and transports them into the flagellum. We suggest that this TOG-domain protein is necessary to efficiently and preemptively increase intra-flagella tubulin levels to offset decreasing IFT cargo at the tip as flagellar assembly progresses.

A Novel Strategy to Uncover Specific Go Terms/phosphorylation Pathways in Phosphoproteomic Data in Arabidopsis Thaliana

Background. Proteins are the workforce of the cell and their phosphorylation status tailors specific responses efficiently. One of the main challenges of phosphoproteomic approaches is to deconvolute biological processes that specifically respond to an experimental query from a list of phosphoproteins. Comparison of the frequency distribution of GO (Gene Ontology) terms in a given phosphoproteome set with that observed in the genome reference set (GenRS) is the most widely used tool to infer biological significance. Yet, this comparison assumes that GO term distribution between the phosphoproteome and the genome are identical. However, this hypothesis has not been tested due to the lack of a comprehensive phosphoproteome database.Results. In this study, we test this hypothesis by constructing three phosphoproteome databases in Arabidopsis thaliana: one based in experimental data (ExpRS), another based in in silico phosphorylation protein prediction (PredRS) and a third that is the union of both (UnRS). Our results show that the three phosphoproteome reference sets show default enrichment of several GO terms compared to GenRS, indicating that GO term distribution in the phosphoproteomes does not match that of the genome. Moreover, these differences overshadow the identification of GO terms that are specifically enriched in a particular condition. To overcome this limitation, we present an additional comparison of the sample of interest with UnRS to uncover GO terms specifically enriched in a particular phosphoproteome experiment. Using this strategy, we found that mRNA splicing and cytoplasmic microtubule compounds are important processes specifically enriched in the phosphoproteome of dark-grown Arabidopsis seedlings. Conclusions. This study provides a novel strategy to uncover GO specific terms in phosphoproteome data of Arabidopsis that could be applied to any other organism. We also highlight the importance of specific phosphorylation pathways that take place during dark-grown Arabidopsis development.

The short flagella 1 (SHF1) gene in Chlamydomonas encodes a Crescerin TOG-domain protein required for late stages of flagellar growth.

Length control of flagella represents a simple and tractable system to investigate the dynamics of organelle size. Models for flagellar length control in the model organism, Chlamydomonas reinhardtii have focused on the length-dependence of the intraflagellar transport (IFT) system which manages the delivery and removal of axonemal subunits at the tip of the flagella. One of these cargoes, tubulin, is the major axonemal subunit, and its frequency of arrival at the tip plays a central role in size control models. However, the mechanisms determining tubulin dynamics at the tip are still poorly understood. We discovered a loss-of-function mutation that leads to shortened flagella, and found that this was an allele of a previously described gene, SHF1, whose molecular identity had not previously been determined. We found that SHF1 encodes a Chlamydomonas ortholog of Crescerin, previously identified as a cilia specific TOG-domain array protein that can bind tubulin via its TOG domains and increase tubulin polymerization rates. In this mutant, flagellar regeneration occurs with the same initial kinetics as wild-type cells, but plateaus at a shorter length. Using a computational model in which the flagellar microtubules are represented by a differential equation for flagellar length combined with a stochastic model for cytoplasmic microtubule dynamics, we found that our experimental results are best described by a model in which Crescerin/SHF1 binds tubulin dimers in the cytoplasm and transports them into the flagellum. We suggest that this TOG-domain protein is necessary to efficiently and preemptively increase intra-flagella tubulin levels to offset decreasing IFT cargo at the tip as flagellar assembly progresses.

High interaction valency ensures cohesion and persistence of a microtubule +TIP body at the plus-end of a single specialized microtubule in yeast

Microtubule plus-end tracking proteins (+TIPs) control microtubule specialization and are as such essential notably during eukaryotic cell division. Here, we investigated interactions and functions of the budding yeast Kar9 network consisting of the core +TIPs components Kar9 (functional homologue of APC, MACF, and SLAIN), Bim1 (orthologue of EB1), and Bik1 (orthologue of CLIP-170). Our data indicate that a redundant, multivalent web of interactions links the three +TIPs together to form a "Kar9 body" at the tip of a single cytoplasmic microtubule. They further suggest that this body is a liquid-like condensate, allowing it to persist on both growing and shrinking microtubule tips, and functions as a mechanical coupling device between microtubules and actin cables during mitosis. Our study underlines the power of dissecting the web of low-affinity interactions driving liquid-liquid phase separation of proteins in order to demonstrate the importance and establish the functional roles of condensation processes in vivo.

Ccdc103 promotes myeloid cell proliferation and migration independent of motile cilia

The pathology of primary ciliary dyskinesia (PCD) is predominantly attributed to impairment of motile cilia. However, PCD patients also have perplexing functional defects in myeloid cells, which lack motile cilia. Here, we show Coiled-coil domain containing protein 103 (CCDC103), one of the genes that when mutated is known to cause PCD, is required for the proliferation and directed migration of myeloid cells. CCDC103 is expressed in human myeloid cells, where it co-localizes with cytoplasmic microtubules. Zebrafish ccdc103/schmalhans (smh) mutants have macrophages and neutrophils with reduced proliferation, abnormally-rounded cell morphology, and an inability to migrate efficiently to the site of sterile wounds, all of which are consistent with a loss of cytoplasmic microtubule stability. Furthermore, we demonstrate that direct interactions between CCDC103 and Sperm associated antigen 6 (SPAG6), which also promotes microtubule stability, are abrogated by CCDC103 mutations from PCD patients, and that spag6 zebrafish mutants recapitulate the myeloid defects observed in smh mutants. In summary, we have illuminated a mechanism, independent of motile cilia, to explain functional defects in myeloid cells from PCD patients.

Naegleria’s mitotic spindles are built from unique tubulins and highlight core spindle features

ABSTRACTNaegleria gruberi is a unicellular eukaryote whose evolutionary distance from animals and fungi has made it useful for developing hypotheses about the last common eukaryotic ancestor. Naegleria amoebae lack a cytoplasmic microtubule cytoskeleton and assemble microtubules only during mitosis, and thus provides a unique system to study the evolution and functional specificity of mitotic tubulins and the resulting spindle. Previous studies showed that Naegleria amoebae express a divergent α-tubulin during mitosis and we now show that Naegleria amoebae express a second mitotic α- and two mitotic β-tubulins. The mitotic tubulins are evolutionarily divergent relative to typical α- and β- tubulins, contain residues that suggest distinct microtubule properties, and may represent drug targets for the “brain-eating amoeba” Naegleria fowleri. Using quantitative light microscopy, we find that Naegleria’s mitotic spindle is a distinctive barrel-like structure built from a ring of microtubule bundles. Similar to those of other species, Naegleria’s spindle is twisted and its length increases during mitosis suggesting that these aspects of mitosis are ancestral features. Because bundle numbers change during metaphase, we hypothesize that the initial bundles represent kinetochore fibers, and secondary bundles function as bridging fibers.

Cytoplasmic Microtubule Organizing Centers Regulate Meiotic Spindle Positioning in Mouse Oocyte

ABSTRACTDuring oocyte meiosis, migration of the spindle and its positioning must be tightly regulated to ensure elimination of the polar bodies and provide developmentally competent euploid eggs. Although the role of F-actin in regulating these critical processes has been studied extensively, little is known whether microtubules (MTs) participate in regulating these processes. Here, we characterize a pool of MTOCs in the oocyte that does not contribute to spindle assembly but instead remains free in the cytoplasm during metaphase I (metaphase cytoplasmic MTOCs; mcMTOCs). In contrast to spindle pole MTOCs, which primarily originate from the perinuclear region in prophase I, the mcMTOCs are found near the cortex of the oocyte. At nuclear envelope breakdown, they exhibit robust nucleation of MTs, which diminishes during polar body extrusion before returning robustly during metaphase II. The asymmetric positioning of the mcMTOCs provides the spindle with a MT-based anchor line to the cortex opposite the site of polar body extrusion. Depletion of mcMTOCs, by laser ablation, or manipulating their numbers, through autophagy inhibition, revealed that the mcMTOCs are required to regulate the timely migration and positioning of the spindle in meiosis. We discuss how forces exerted by F-actin in mediating movement of the spindle to the oocyte cortex are balanced by MT-mediated forces from the mcMTOCs to ensure spindle positioning and timely spindle migration.