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1.  Coiled-Coil Proteins Facilitated the Functional Expansion of the Centrosome 
PLoS Computational Biology  2014;10(6):e1003657.
Repurposing existing proteins for new cellular functions is recognized as a main mechanism of evolutionary innovation, but its role in organelle evolution is unclear. Here, we explore the mechanisms that led to the evolution of the centrosome, an ancestral eukaryotic organelle that expanded its functional repertoire through the course of evolution. We developed a refined sequence alignment technique that is more sensitive to coiled coil proteins, which are abundant in the centrosome. For proteins with high coiled-coil content, our algorithm identified 17% more reciprocal best hits than BLAST. Analyzing 108 eukaryotic genomes, we traced the evolutionary history of centrosome proteins. In order to assess how these proteins formed the centrosome and adopted new functions, we computationally emulated evolution by iteratively removing the most recently evolved proteins from the centrosomal protein interaction network. Coiled-coil proteins that first appeared in the animal–fungi ancestor act as scaffolds and recruit ancestral eukaryotic proteins such as kinases and phosphatases to the centrosome. This process created a signaling hub that is crucial for multicellular development. Our results demonstrate how ancient proteins can be co-opted to different cellular localizations, thereby becoming involved in novel functions.
Author Summary
The centrosome helps cells to divide, and is important for the development of animals. It has its evolutionary origins in the basal body, which was present in the last common ancestor of all eukaryotes. Here, we study how the evolution of novel proteins helped the formation of the centrosome. Coiled-coil proteins are important for the function of the centrosome. But, they have repeating patterns that can confuse existing methods for finding related proteins. We refined these methods by adjusting for the special properties of the coiled-coil regions. This enabled us to find more distant relatives of centrosomal proteins. We then tested how novel proteins affect the protein interaction network of the centrosome. We did this by removing the most novel proteins step by step. At each stage, we observed how the remaining proteins are connected to the centriole, the core of the centrosome. We found that coiled-coil proteins that first occurred in the ancestor of fungi and animals help to recruit older proteins. By being recruited to the centrosome, these older proteins acquired new functions. We thus now have a clearer picture of how the centrosome became such an important part of animal cells.
doi:10.1371/journal.pcbi.1003657
PMCID: PMC4046923  PMID: 24901223
2.  The human cap-binding complex is functionally connected to the nuclear RNA exosome 
Nature structural & molecular biology  2013;20(12):1367-1376.
Nuclear processing and quality control of eukaryotic RNA is mediated by the RNA exosome, which is regulated by accessory factors. However, the mechanism of exosome recruitment to its ribonucleoprotein (RNP) targets remains poorly understood. Here we disclose a physical link between the human exosome and the cap-binding complex (CBC). The CBC associates with the ARS2 protein to form CBC-ARS2 (CBCA), and then further connects together with the ZC3H18 protein to the nuclear exosome targeting (NEXT) complex, forming CBC-NEXT (CBCN). RNA immunoprecipitation using CBCN factors as well as the analysis of combinatorial depletion of CBCN and exosome components underscore the functional relevance of CBC-exosome bridging at the level of target RNA. Specifically, CBCA suppresses read-through products of several RNA families by promoting their transcriptional termination. We suggest that the RNP 5′cap links transcription termination to exosomal RNA degradation via CBCN.
doi:10.1038/nsmb.2703
PMCID: PMC3923317  PMID: 24270879
3.  Rheotaxis facilitates upstream navigation of mammalian sperm cells 
eLife  2014;3:e02403.
A major puzzle in biology is how mammalian sperm maintain the correct swimming direction during various phases of the sexual reproduction process. Whilst chemotaxis may dominate near the ovum, it is unclear which cues guide spermatozoa on their long journey towards the egg. Hypothesized mechanisms range from peristaltic pumping to temperature sensing and response to fluid flow variations (rheotaxis), but little is known quantitatively about them. We report the first quantitative study of mammalian sperm rheotaxis, using microfluidic devices to investigate systematically swimming of human and bull sperm over a range of physiologically relevant shear rates and viscosities. Our measurements show that the interplay of fluid shear, steric surface-interactions, and chirality of the flagellar beat leads to stable upstream spiralling motion of sperm cells, thus providing a generic and robust rectification mechanism to support mammalian fertilisation. A minimal mathematical model is presented that accounts quantitatively for the experimental observations.
DOI: http://dx.doi.org/10.7554/eLife.02403.001
eLife digest
A sperm cell must complete a long and taxing journey to stand a chance of fertilising an egg cell. This quest covers a distance that is thousands of times longer than the length of a sperm cell. It also passes through the diverse environments of the cervix, the uterus and, finally, the oviduct, where there might be an egg to fertilise. How the sperm cells manage to stay on course over this distance is a mystery, although it has been suggested that many different factors, including chemical signals and fluid flow, are involved.
The fluids that the sperm cells travel through are not static. Evidence suggests that contractions of the cervix and uterus help to pump sperm cells along the first part of their journey. However, mucus flows out of the oviduct in the opposite direction to way the sperm cells need to go.
Sperm cells mostly move along the walls of the cervix, uterus, and oviduct. This means that sperm cells must contend with two properties of the fluids they travel through—the viscosity (or ‘thickness’) of the fluid, and the fact that different parts of the fluid will flow at different speeds, depending on how close it is to the wall (‘shear flow’).
Kantsler et al. have now used a technique called microfluidics—which involves forcing tiny amounts of liquid to flow through very narrow channels—to study how the movement of human and bull sperm cells along a surface is affected by the viscosity and flow rate of the fluid they are swimming through. The sperm cells were found to swim upstream, moving along the walls of the channels in a spiral movement. This is likely to help the sperm cells to find the egg, because spiralling around the oviduct will increase the chances of meeting the egg.
Kantsler et al. also built a mathematical model that describes how the sperm cells move. Although further work is needed to better understand the role played by chemical signals, understanding how fluid flow and viscosity influence sperm cells could lead to more effective artificial insemination techniques.
DOI: http://dx.doi.org/10.7554/eLife.02403.002
doi:10.7554/eLife.02403
PMCID: PMC4031982  PMID: 24867640
sperm; rheotaxis; fertilization; human; other
4.  Ki-67 is a PP1-interacting protein that organises the mitotic chromosome periphery 
eLife  2014;3:e01641.
When the nucleolus disassembles during open mitosis, many nucleolar proteins and RNAs associate with chromosomes, establishing a perichromosomal compartment coating the chromosome periphery. At present nothing is known about the function of this poorly characterised compartment. In this study, we report that the nucleolar protein Ki-67 is required for the assembly of the perichromosomal compartment in human cells. Ki-67 is a cell-cycle regulated protein phosphatase 1-binding protein that is involved in phospho-regulation of the nucleolar protein B23/nucleophosmin. Following siRNA depletion of Ki-67, NIFK, B23, nucleolin, and four novel chromosome periphery proteins all fail to associate with the periphery of human chromosomes. Correlative light and electron microscopy (CLEM) images suggest a near-complete loss of the entire perichromosomal compartment. Mitotic chromosome condensation and intrinsic structure appear normal in the absence of the perichromosomal compartment but significant differences in nucleolar reassembly and nuclear organisation are observed in post-mitotic cells.
DOI: http://dx.doi.org/10.7554/eLife.01641.001
eLife digest
The genetic information of an organism is found in the nucleus of each cell in the form of DNA organised into chromosomes. The exact structure of those chromosomes changes as the cell moves through the different stages of the cell division cycle. During the stage called mitosis, where the DNA of a cell (which has previously been duplicated) is shared into two daughter cells, the chromosomes become tightly packed structures that can be readily moved through the cytoplasm. Since the late nineteenth century, it has been known that a layer of proteins, called the perichromosomal layer, coats the condensed chromosomes. However, virtually nothing was known about the role this layer performs.
One of the first proteins to join the perichromosomal layer after mitosis begins is called Ki-67. This is only found in the cell nucleus when a cell is actively growing and dividing, and so is widely used as a marker in experiments investigating these processes: for example, Ki-67 is used to detect growing tumour cells amongst the normal cells in tissues of the body, and to measure the effectiveness of drugs designed to stop the growth of tumours. Again, however, little is known about what Ki-67 actually does.
Booth et al. now reveal that when Ki-67 is not present in a cell, chromosomes do not have a perichromosomal layer—or at best, have a small remnant of one. This allowed Booth et al. to investigate the role of the perichromosomal layer as well. When the chromosomes first go through mitosis without a perichromosomal layer, no changes to the shape or the behaviour of the chromosomes are seen. However, the new nuclei are smaller than normal and their contents are arranged differently. This causes problems with the ability of daughter cells to synthesise protein building blocks and leads to an increased rate of spontaneous cell death when daughter cells try to undergo the next mitosis. Further research is needed to understand why this happens.
DOI: http://dx.doi.org/10.7554/eLife.01641.002
doi:10.7554/eLife.01641
PMCID: PMC4032110  PMID: 24867636
chromosomes; mitosis; phosphatases; nucleolus; human
5.  Encouraging innovation 
Molecular Biology of the Cell  2014;25(4):427-428.
Innovation is central to the scientific endeavor, and yet the current system of funding in the United States discourages innovation, especially in the young. Subtle alterations to the funding system, guided in part by the success of the European Research Council, could have major effects on encouraging innovation.
doi:10.1091/mbc.E13-10-0596
PMCID: PMC3923634  PMID: 24525947
6.  The GTPase IFT27 is involved in both anterograde and retrograde intraflagellar transport 
eLife  2014;3:e02419.
The construction of cilia and flagella depends on intraflagellar transport (IFT), the bidirectional movement of two protein complexes (IFT-A and IFT-B) driven by specific kinesin and dynein motors. IFT-B and kinesin are associated to anterograde transport whereas IFT-A and dynein participate to retrograde transport. Surprisingly, the small GTPase IFT27, a member of the IFT-B complex, turns out to be essential for retrograde cargo transport in Trypanosoma brucei. We reveal that this is due to failure to import both the IFT-A complex and the IFT dynein into the flagellar compartment. To get further molecular insight about the role of IFT27, GDP- or GTP-locked versions were expressed in presence or absence of endogenous IFT27. The GDP-locked version is unable to enter the flagellum and to interact with other IFT-B proteins and its sole expression prevents flagellum formation. These findings demonstrate that a GTPase-competent IFT27 is required for association to the IFT complex and that IFT27 plays a role in the cargo loading of the retrograde transport machinery.
DOI: http://dx.doi.org/10.7554/eLife.02419.001
eLife digest
Long, thin structures called cilia and flagella are found on the surface of many cells, and perform a range of roles, including propelling the cells around or sensing changes in the surrounding environment. A process called intraflagellar transport (IFT for short) is responsible for flagellum construction in eukaryotic cells. Protein complexes called IFT trains carry the building blocks that make up flagella along microtubule ‘tracks’ between the base and the tip of a flagellum.
IFT trains are made from two different protein complexes called IFT-A and IFT-B, which are dragged by various molecular motors. The IFT-B complex is necessary for the train to move towards the tip of the flagellum, and so enables the flagellum to grow. The IFT-A protein complex is required to recycle the train back towards the base of the flagellum.
Huet et al. examined the role that a protein called IFT27 plays in intraflagellar transport. IFT27 is part of the IFT-B complex, and so it was thought to only affect how flagella grow. However, short flagella still grow when IFT27 is absent, but they are filled with IFT trains that are not able to reverse back from the tip. Huet et al. reveal that the IFT-A complex and the molecular motor that is essential for reversing the train are not transported into the flagellum if IFT27 is not present. This is therefore an unusual case of an IFT-B protein affecting the IFT-A complex and the transport back to the base.
IFT27 also affects how the IFT-B complex forms. ITF27 can bind to some small molecules, which can switch the protein ‘on’ or ‘off’. Huet et al. found that when IFT27 is switched off it is not transported into flagella, and also cannot bind to some of the other proteins in the IFT-B complex. This means that if IFT27 is locked in an inactive state, the IFT-B complex does not form, and a flagellum cannot grow. Therefore, activated IFT27 is needed for putting together the IFT train and to ensure its movement in either direction along the microtubule tracks.
DOI: http://dx.doi.org/10.7554/eLife.02419.002
doi:10.7554/eLife.02419
PMCID: PMC4003483  PMID: 24843028
Trypanosoma brucei; flagellum; small GTPase; IFT; other
7.  Structural basis for the assembly of the mitotic motor Kinesin-5 into bipolar tetramers 
eLife  2014;3:e02217.
Chromosome segregation during mitosis depends upon Kinesin-5 motors, which display a conserved, bipolar homotetrameric organization consisting of two motor dimers at opposite ends of a central rod. Kinesin-5 motors crosslink adjacent microtubules to drive or constrain their sliding apart, but the structural basis of their organization is unknown. In this study, we report the atomic structure of the bipolar assembly (BASS) domain that directs four Kinesin-5 subunits to form a bipolar minifilament. BASS is a novel 26-nm four-helix bundle, consisting of two anti-parallel coiled-coils at its center, stabilized by alternating hydrophobic and ionic four-helical interfaces, which based on mutagenesis experiments, are critical for tetramerization. Strikingly, N-terminal BASS helices bend as they emerge from the central bundle, swapping partner helices, to form dimeric parallel coiled-coils at both ends, which are offset by 90°. We propose that BASS is a mechanically stable, plectonemically-coiled junction, transmitting forces between Kinesin-5 motor dimers during microtubule sliding.
DOI: http://dx.doi.org/10.7554/eLife.02217.001
eLife digest
Successful cell division requires copies of the chromosomes containing the genetic material of a cell to be accurately copied and then separated so that when a cell divides, each new daughter cell contains exactly one copy of each chromosome. If this does not happen, the cell may malfunction or die.
To separate the duplicated chromosomes, a biological machine called the mitotic spindle forms inside the cell. This has two poles, one at each end, with each pole being responsible for gathering together the chromosomes for delivery to each of the daughter cells. Large numbers of long, thin protein tubes called microtubules extend out of each pole. Some microtubules attach to the chromosomes, whilst others are responsible for pushing apart the two poles—and the chromosomes attached to them—to the opposite sides of the cell before it divides.
To move the poles, motor proteins slide pairs of microtubules that are attached to opposite poles over each other. The Kinesin-5 family of motor proteins is particularly important for mitosis, because it is essential for forming the mitotic spindle and for making it work correctly. These motors assemble into motile machines that can apply a force to both of the microtubules in a sliding pair at the same time because they contain motor units at each end connected by a central rod.
The structure of this central rod is crucial for the successful operation of Kinesin-5. Scholey, Nithianantham et al. have now worked out the structure of a region of this filament called the bipolar assembly, or BASS domain. This structure is more complicated than expected: it contains four helixes made of protein that are all intertwined with each other.
In addition, Scholey, Nithianantham et al. found two ‘molecular pockets’ that small molecules can access. By entering the pockets, the molecules could disrupt the structure of the BASS domain, and consequently prevent Kinesin-5 from forming the dual-ended machines required to work properly. As Kinesin-5 is required to build the mitotic spindle, this would interfere with cell division. Targeting molecules into these pockets could therefore potentially form part of an anti-cancer therapy, preventing the rapid cell divisions behind the spread of the disease.
DOI: http://dx.doi.org/10.7554/eLife.02217.002
doi:10.7554/eLife.02217
PMCID: PMC3978770  PMID: 24714498
mitosis; Kinesin-5; microtubule; motor protein; coiled-coil; X-ray structure; D. melanogaster
8.  A genome scale resource for in vivo tag-based protein function exploration in C. elegans 
Cell  2012;150(4):855-866.
Understanding the in vivo dynamics of protein localization and their physical interactions is important for many problems in Biology. To enable systematic protein function interrogation in a multicelluar context, we built a genome-scale transgenic platform for in vivo expression of fluorescent and affinity tagged proteins in Caenorhabditis elegans under endogenous cis regulatory control. The platform combines computer-assisted transgene design, massively parallel DNA engineering and next generation sequencing to generate a resource of 14637 genomic DNA transgenes, which covers 73% of the proteome. The multipurpose tag used allows any protein of interest to be localized in vivo or affinity purified using standard tag-based assays. We illustrate the utility of the resource by systematic chromatin immunopurification and automated 4D imaging, which produced detailed DNA binding and cell/tissue distribution maps for key transcription factor proteins
doi:10.1016/j.cell.2012.08.001
PMCID: PMC3979301  PMID: 22901814
9.  C11ORF24 Is a Novel Type I Membrane Protein That Cycles between the Golgi Apparatus and the Plasma Membrane in Rab6-Positive Vesicles 
PLoS ONE  2013;8(12):e82223.
The Golgi apparatus is an intracellular compartment necessary for post-translational modification, sorting and transport of proteins. It plays a key role in mitotic entry through the Golgi mitotic checkpoint. In order to identify new proteins involved in the Golgi mitotic checkpoint, we combine the results of a knockdown screen for mitotic phenotypes and a localization screen. Using this approach, we identify a new Golgi protein C11ORF24 (NP_071733.1). We show that C11ORF24 has a signal peptide at the N-terminus and a transmembrane domain in the C-terminal region. C11ORF24 is localized on the Golgi apparatus and on the trans-Golgi network. A large part of the protein is present in the lumen of the Golgi apparatus whereas only a short tail extends into the cytosol. This cytosolic tail is well conserved in evolution. By FRAP experiments we show that the dynamics of C11ORF24 in the Golgi membrane are coherent with the presence of a transmembrane domain in the protein. C11ORF24 is not only present on the Golgi apparatus but also cycles to the plasma membrane via endosomes in a pH sensitive manner. Moreover, via video-microscopy studies we show that C11ORF24 is found on transport intermediates and is colocalized with the small GTPase RAB6, a GTPase involved in anterograde transport from the Golgi to the plasma membrane. Knocking down C11ORF24 does not lead to a mitotic phenotype or an intracellular transport defect in our hands. All together, these data suggest that C11ORF24 is present on the Golgi apparatus, transported to the plasma membrane and cycles back through the endosomes by way of RAB6 positive carriers.
doi:10.1371/journal.pone.0082223
PMCID: PMC3846831  PMID: 24312644
10.  Live-cell imaging RNAi screen identifies PP2A–B55α and importin-β1 as key mitotic exit regulators in human cells 
Nature cell biology  2010;12(9):10.1038/ncb2092.
When vertebrate cells exit mitosis various cellular structures are re-organized to build functional interphase cells1. This depends on Cdk1 (cyclin dependent kinase 1) inactivation and subsequent dephosphorylation of its substrates2–4. Members of the protein phosphatase 1 and 2A (PP1 and PP2A) families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit5,6, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we use a live-cell imaging assay and RNAi knockdown to screen a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identify a trimeric PP2A–B55α complex as a key factor in mitotic spindle breakdown and postmitotic reassembly of the nuclear envelope, Golgi apparatus and decondensed chromatin. Using a chemically induced mitotic exit assay, we find that PP2A–B55α functions downstream of Cdk1 inactivation. PP2A–B55α isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate, histone H1, and was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor importin-β1, and RNAi depletion of importin-β1 delayed mitotic exit synergistically with PP2A–B55α. This demonstrates that PP2A–B55α and importin-β1 cooperate in the regulation of postmitotic assembly mechanisms in human cells.
doi:10.1038/ncb2092
PMCID: PMC3839080  PMID: 20711181
11.  A Systematic Mammalian Genetic Interaction Map Reveals Pathways Underlying Ricin Susceptibility 
Cell  2013;152(4):909-922.
SUMMARY
Genetic interaction (GI) maps, comprising pairwise measures of how strongly the function of one gene depends on the presence of a second, have enabled the systematic exploration of gene function in microorganisms. Here, we present a two-stage strategy to construct high-density GI maps in mammalian cells. First, we use ultra-complex pooled shRNA libraries (25 shRNAs/gene) to identify high-confidence hit genes for a given phenotype and effective shRNAs. We then construct double-shRNA libraries from these to systematically measure GIs between hits. A GI map focused on ricin susceptibility broadly recapitulates known pathways and provides many unexpected insights. These include a non-canonical role for COPI, a novel protein complex (SRIC) affecting toxin clearance, a specialized role for the ribosomal protein RPS25, and functionally distinct mammalian TRAPP complexes. The ability to rapidly generate mammalian GI maps provides a potentially transformative tool for defining gene function and designing combination therapies based on synergistic pairs.
doi:10.1016/j.cell.2013.01.030
PMCID: PMC3652613  PMID: 23394947
12.  APC15 mediates CDC20 auto-ubiquitylation by APC/CMCC and MCC disassembly 
Nature structural & molecular biology  2012;19(11):1116-1123.
The anaphase-promoting complex/cyclosome bound to CDC20 (APC/CCDC20) initiates anaphase by ubiquitylating B-type cyclins and securin. During chromosome bi-orientation, CDC20 assembles with MAD2, BUBR1 and BUB3 into a mitotic checkpoint complex (MCC) which inhibits substrate recruitment to the APC/C. APC/C activation depends on MCC disassembly, which has been proposed to require CDC20 auto-ubiquitylation. Here we characterized APC15, a human APC/C subunit related to yeast Mnd2. APC15 is located near APC/C’s MCC binding site, is required for APC/CMCC-dependent CDC20 auto-ubiquitylation and degradation, and for timely anaphase initiation, but is dispensable for substrate ubiquitylation by APC/CCDC20 and APC/CCDH1. Our results support the view that MCC is continuously assembled and disassembled to enable rapid activation of APC/CCDC20 and that CDC20 auto-ubiquitylation promotes MCC disassembly. We propose that APC15 and Mnd2 negatively regulate APC/C coactivators, and report the first generation of recombinant human APC/C.
doi:10.1038/nsmb.2412
PMCID: PMC3498062  PMID: 23007861
14.  One-step purification of assembly-competent tubulin from diverse eukaryotic sources 
Molecular Biology of the Cell  2012;23(22):4393-4401.
A method is presented that allows rapid and efficient purification of native, active tubulin from a variety of species and tissue sources by affinity chromatography. It eliminates the need to use heterologous systems for the study of microtubule-associated proteins and motor proteins, which has been a major issue in microtubule-related research.
We have developed a protocol that allows rapid and efficient purification of native, active tubulin from a variety of species and tissue sources by affinity chromatography. The affinity matrix comprises a bacterially expressed, recombinant protein, the TOG1/2 domains from Saccharomyces cerevisiae Stu2, covalently coupled to a Sepharose support. The resin has a high capacity to specifically bind tubulin from clarified crude cell extracts, and, after washing, highly purified tubulin can be eluted under mild conditions. The eluted tubulin is fully functional and can be efficiently assembled into microtubules. The method eliminates the need to use heterologous systems for the study of microtubule-associated proteins and motor proteins, which has been a major issue in microtubule-related research.
doi:10.1091/mbc.E12-06-0444
PMCID: PMC3496613  PMID: 22993214
15.  Sds22 and Repo-Man stabilize chromosome segregation by counteracting Aurora B on anaphase kinetochores 
The Journal of Cell Biology  2012;198(2):173-183.
Repo-Man and Sds22 counteract Aurora B phosphorylation of Dsn1 and thus regulate the kinetochore–microtubule interface during anaphase.
During mitotic spindle assembly, Aurora B kinase is part of an error correction mechanism that detaches microtubules from kinetochores that are under low mechanical tension. During anaphase, however, kinetochore–microtubule attachments must be maintained despite a drop of tension after removal of sister chromatid cohesion. Consistent with this requirement, Aurora B relocates away from chromosomes to the central spindle at the metaphase–anaphase transition. By ribonucleic acid interference screening using a phosphorylation biosensor, we identified two PP1-targeting subunits, Sds22 and Repo-Man, which counteracted Aurora B–dependent phosphorylation of the outer kinetochore component Dsn1 during anaphase. Sds22 or Repo-Man depletion induced transient pauses during poleward chromosome movement and a high incidence of chromosome missegregation. Thus, our study identifies PP1-targeting subunits that regulate the microtubule–kinetochore interface during anaphase for faithful chromosome segregation.
doi:10.1083/jcb.201112112
PMCID: PMC3410419  PMID: 22801782
16.  BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures 
Molecular Biology of the Cell  2012;23(21):4226-4241.
This study dissects the recruitment of dynein and dynactin to cargo by a conserved motor adaptor BICD2. It is shown that dynein, dynactin, and BICD2 form a triple complex in vitro and in vivo. Investigation of the properties of this complex by direct visualization of dynein in live cells shows that BICD2-induced dynein transport requires LIS1.
Cytoplasmic dynein is the major microtubule minus-end–directed cellular motor. Most dynein activities require dynactin, but the mechanisms regulating cargo-dependent dynein–dynactin interaction are poorly understood. In this study, we focus on dynein–dynactin recruitment to cargo by the conserved motor adaptor Bicaudal D2 (BICD2). We show that dynein and dynactin depend on each other for BICD2-mediated targeting to cargo and that BICD2 N-terminus (BICD2-N) strongly promotes stable interaction between dynein and dynactin both in vitro and in vivo. Direct visualization of dynein in live cells indicates that by itself the triple BICD2-N–dynein–dynactin complex is unable to interact with either cargo or microtubules. However, tethering of BICD2-N to different membranes promotes their microtubule minus-end–directed motility. We further show that LIS1 is required for dynein-mediated transport induced by membrane tethering of BICD2-N and that LIS1 contributes to dynein accumulation at microtubule plus ends and BICD2-positive cellular structures. Our results demonstrate that dynein recruitment to cargo requires concerted action of multiple dynein cofactors.
doi:10.1091/mbc.E12-03-0210
PMCID: PMC3484101  PMID: 22956769
17.  GTSE1 Is a Microtubule Plus-End Tracking Protein That Regulates EB1-Dependent Cell Migration 
PLoS ONE  2012;7(12):e51259.
The regulation of cell migration is a highly complex process that is often compromised when cancer cells become metastatic. The microtubule cytoskeleton is necessary for cell migration, but how microtubules and microtubule-associated proteins regulate multiple pathways promoting cell migration remains unclear. Microtubule plus-end binding proteins (+TIPs) are emerging as important players in many cellular functions, including cell migration. Here we identify a +TIP, GTSE1, that promotes cell migration. GTSE1 accumulates at growing microtubule plus ends through interaction with the EB1+TIP. The EB1-dependent +TIP activity of GTSE1 is required for cell migration, as well as for microtubule-dependent disassembly of focal adhesions. GTSE1 protein levels determine the migratory capacity of both nontransformed and breast cancer cell lines. In breast cancers, increased GTSE1 expression correlates with invasive potential, tumor stage, and time to distant metastasis, suggesting that misregulation of GTSE1 expression could be associated with increased invasive potential.
doi:10.1371/journal.pone.0051259
PMCID: PMC3517537  PMID: 23236459
18.  Stoichiometry of chromatin-associated protein complexes revealed by label-free quantitative mass spectrometry-based proteomics 
Nucleic Acids Research  2012;41(1):e28.
Many cellular proteins assemble into macromolecular protein complexes. The identification of protein–protein interactions and quantification of their stoichiometry is therefore crucial to understand the molecular function of protein complexes. Determining the stoichiometry of protein complexes is usually achieved by mass spectrometry-based methods that rely on introducing stable isotope-labeled reference peptides into the sample of interest. However, these approaches are laborious and not suitable for high-throughput screenings. Here, we describe a robust and easy to implement label-free relative quantification approach that combines the detection of high-confidence protein–protein interactions with an accurate determination of the stoichiometry of the identified protein–protein interactions in a single experiment. We applied this method to two chromatin-associated protein complexes for which the stoichiometry thus far remained elusive: the MBD3/NuRD and PRC2 complex. For each of these complexes, we accurately determined the stoichiometry of the core subunits while at the same time identifying novel interactors and their stoichiometry.
doi:10.1093/nar/gks941
PMCID: PMC3592467  PMID: 23066101
19.  A High-Resolution C. elegans Essential Gene Network Based on Phenotypic Profiling of a Complex Tissue 
Cell  2011;145(3):470-482.
SUMMARY
High-content screening for gene profiling has generally been limited to single cells. Here, we explore an alternative approach—profiling gene function by analyzing effects of gene knockdowns on the architecture of a complex tissue in a multicellular organism. We profile 554 essential C. elegans genes by imaging gonad architecture and scoring 94 phenotypic features. To generate a reference for evaluating methods for network construction, genes were manually partitioned into 102 phenotypic classes, predicting functions for uncharacterized genes across diverse cellular processes. Using this classification as a benchmark, we developed a robust computational method for constructing gene networks from high-content profiles based on a network context-dependent measure that ranks the significance of links between genes. Our analysis reveals that multi-parametric profiling in a complex tissue yields functional maps with a resolution similar to genetic interaction-based profiling in unicellular eukaryotes—pinpointing subunits of macromolecular complexes and components functioning in common cellular processes.
doi:10.1016/j.cell.2011.03.037
PMCID: PMC3086541  PMID: 21529718
20.  Whither systems biology 
Cell biologists are interested in how complexity arises from the interaction of different molecules. However, cells are many orders of magnitude larger than the protein-binding interfaces. To bridge these vast difference in scales, biologists construct hierarchies of organization of cellular structures. I describe how systems biology provides an approach to bridge these different scales.
doi:10.1098/rstb.2011.0074
PMCID: PMC3203457  PMID: 22084389
systems biology; modelling; cell cycle
21.  PAR proteins diffuse freely across the anterior–posterior boundary in polarized C. elegans embryos 
The Journal of Cell Biology  2011;193(3):583-594.
FRAP reveals that a stable PAR boundary requires balancing diffusive flux of PAR proteins between domains with spatial differences in PAR protein membrane affinities.
Polarization of cells by PAR proteins requires the segregation of antagonistic sets of proteins into two mutually exclusive membrane-associated domains. Understanding how nanometer scale interactions between individual PAR proteins allow spatial organization across cellular length scales requires determining the kinetic properties of PAR proteins and how they are modified in space. We find that PAR-2 and PAR-6, which localize to opposing PAR domains, undergo exchange between well mixed cytoplasmic populations and laterally diffusing membrane-associated states. Domain maintenance does not involve diffusion barriers, lateral sorting, or active transport. Rather, both PAR proteins are free to diffuse between domains, giving rise to a continuous boundary flux because of lateral diffusion of molecules down the concentration gradients that exist across the embryo. Our results suggest that the equalizing effects of lateral diffusion are countered by actin-independent differences in the effective membrane affinities of PAR proteins between the two domains, which likely depend on the ability of each PAR species to locally modulate the membrane affinity of opposing PAR species within its domain. We propose that the stably polarized embryo reflects a dynamic steady state in which molecules undergo continuous diffusion between regions of net association and dissociation.
doi:10.1083/jcb.201011094
PMCID: PMC3087016  PMID: 21518794
22.  Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes 
Nature  2010;464(7289):721-727.
Despite our rapidly growing knowledge about the human genome, we do not know all of the genes required for some of the most basic functions of life. To start to fill this gap we developed a high-throughput phenotypic screening platform combining potent gene silencing by RNA interference, time-lapse microscopy and computational image processing. We carried out a genome-wide phenotypic profiling of each of the ~21,000 human protein-coding genes by two-day live imaging of fluorescently labelled chromosomes. Phenotypes were scored quantitatively by computational image processing, which allowed us to identify hundreds of human genes involved in diverse biological functions including cell division, migration and survival. As part of the Mitocheck consortium, this study provides an in-depth analysis of cell division phenotypes and makes the entire high-content data set available as a resource to the community.
doi:10.1038/nature08869
PMCID: PMC3108885  PMID: 20360735
23.  Systematic Characterization of Human Protein Complexes Identifies Chromosome Segregation Proteins 
Science (New York, N.Y.)  2010;328(5978):593-599.
Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference (RNAi) screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization and tandem affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex (APC/C) and the γ-tubulin ring complex (γ-TuRC), large complexes which are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high throughput follow-up analyses of phenotypic screens in mammalian cells.
doi:10.1126/science.1181348
PMCID: PMC2989461  PMID: 20360068
24.  The RSA complex targets Protein Phosphatase 2A to centrosomes and regulates mitotic spindle assembly in C. elegans 
Cell  2007;128(1):115-127.
Microtubule behavior changes during the cell cycle and during spindle assembly. However it remains unclear how these changes are regulated and coordinated. We describe a complex that targets the Protein Phosphatase 2A holoenzyme (PP2A) to centrosomes in C. elegans embryos. This complex includes RSA-1, a targeting subunit for PP2A, and RSA-2, a protein that binds and recruits RSA-1 to centrosomes. In contrast to the multiple functions of the PP2A catalytic subunit, RSA-1 and RSA-2 are specifically required for microtubule outgrowth from centrosomes and for spindle assembly. The centrosomally localized RSA-PP2A complex mediates these functions in part by regulating two critical mitotic effectors: the microtubule destabilizer KLP-7 and the C. elegans regulator of spindle assembly TPXL-1. Therefore, by recruiting the PP2A catalytic subunit to centrosomes, the RSA complex regulates a subset of PP2A functions in order to coordinate microtubule outgrowth from centrosomes and microtubule stability in the forming mitotic spindle.
doi:10.1016/j.cell.2006.10.050
PMCID: PMC2987564  PMID: 17218259
25.  Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions 
The Journal of Cell Biology  2010;189(4):739-754.
QUBIC, a specific and highly sensitive method for detection of protein–protein interactions, is used to identify new partners for the mitotic spindle components pericentrin and TACC3.
Protein interactions are involved in all cellular processes. Their efficient and reliable characterization is therefore essential for understanding biological mechanisms. In this study, we show that combining bacterial artificial chromosome (BAC) TransgeneOmics with quantitative interaction proteomics, which we call quantitative BAC–green fluorescent protein interactomics (QUBIC), allows specific and highly sensitive detection of interactions using rapid, generic, and quantitative procedures with minimal material. We applied this approach to identify known and novel components of well-studied complexes such as the anaphase-promoting complex. Furthermore, we demonstrate second generation interaction proteomics by incorporating directed mutational transgene modification and drug perturbation into QUBIC. These methods identified domain/isoform-specific interactors of pericentrin- and phosphorylation-specific interactors of TACC3, which are necessary for its recruitment to mitotic spindles. The scalability, simplicity, cost effectiveness, and sensitivity of this method provide a basis for its general use in small-scale experiments and in mapping the human protein interactome.
doi:10.1083/jcb.200911091
PMCID: PMC2872919  PMID: 20479470

Results 1-25 (57)