Journal : Motors bring genes together

Published online 7 April 2008
doi:10.1083/jcb.1812rr3
The Journal of Cell Biology
© The Rockefeller University Press, 0021-9525 $30.00

Motors bring genes together

Genes on chromosome 2 (red) and 21 (green) come together in the presence of estrogen (bottom). Rosenfeld/Elsevier

Nuclear motors rearrange chromosomes to enhance estrogen-driven transcription, according to Esperanza Nunez, Michael Rosenfeld, Xiang-Dong Fu (University of California at San Diego, CA), and colleagues.

Estrogen-responsive enhancers occur throughout the genome, often long distances from the genes whose transcription they enhance. To determine how these enhancers and their bound estrogen receptors are brought to their gene targets, the authors used FISH to follow their movements. Under the influence of estrogen, two genes on chromosomes 2 and 21 formed 2–21 pairs or tetrads of all four alleles. The team never observed 2–2 or 21–21 pairings. The factors that cause these specific pairings, and prevent others, are unknown.

Chromosomes usually came together within five minutes of estrogen exposure. The movements required cytoskeletal elements and their motors; blocking myosin or the polymerization of nuclear actin abolished the interactions and reduced gene expression. So did blocking dynein light chain 1, which is known to bind to the estrogen receptor. The authors suggest that dynein links the actin to the DNA-bound estrogen receptor.

Other estrogen-regulated gene sets converged in other nuclear locations, each the site of an RNA-processing nuclear speckle. When the authors knocked down a demethylase that is required for estrogen-dependent gene activation, genes came together but did not bind to the speckle. Gene expression from interacting alleles was much higher than for noninteracting ones, indicating that their linkages increase transcription rates.

Nunez, E., et al. 2008. Cell. 132:996–1010. [Abstract]

Richard Robinson

rrobinson@nasw.org

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Journal : MukBs cooperate to loop DNA

Published online 7 April 2008
doi:10.1083/jcb.1812rr2
The Journal of Cell Biology
© The Rockefeller University Press, 0021-9525 $30.00

MukBs cooperate to loop DNA

When it comes to corralling DNA, MukB is the little condensin that could, say Yuanbo Cui, Aoya Petrushenko, and Valentin Rybenkov (University of Oklahoma, Norman, OK).

Small in size but with a big effect, condensins are believed to stabilize large DNA loops, thereby giving order to an otherwise tangled mess of chromatin. To investigate the mechanics of the E. coli condensin MukB, the authors stretched DNA between a glass capillary and a magnetic bead and monitored MukB-induced DNA condensation.

The DNA compacted in a series of discrete steps, indicating that multiple MukBs are at work. The process could be reversed by applying excess force in the opposite direction, but the longer a condensed complex was allowed to sit, the more force was required, suggesting that MukBs link up over time.

A small decrease in MukB concentration led to a dramatic decline in condensation rate. The slowed rate was primarily due to a longer lag before condensation began, which indicates that multiple MukBs must first come together in a nucleation step. ATP stimulated this nucleation but had no subsequent effect, and faster hydrolysis did not lead to faster nucleation. ATP thus seems to be a structural, rather than energetic, component.

According to Rybenkov, MukBs are acting as clamps to hold loops together. “I think we now have a biochemical explanation for how stable loops appear in such a big unruly molecule as DNA,” he says.

Cui, Y., et al. 2008. Nat. Struct. Mol. Biol. doi:10.1038/nsmb.1410. [Abstract]

Richard Robinson

rrobinson@nasw.org

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Journal : Pack loose, grow long

Published online 7 April 2008
doi:10.1083/jcb.1812rr1
The Journal of Cell Biology
© The Rockefeller University Press, 0021-9525 $30.00

Pack loose, grow long

MSP filaments pack more loosely as they elongate away from the site of polymerization (top). Roberts/NAS

Longer cytoskeletal rods have more empty space between them, and that space helps drive amoeboid cell motility, say Thomas Roberts (Florida State University, Tallahassee, FL) and colleagues.

Theories of amoeboid-like cell movement have largely relied on cytoskeleton polymerization to explain membrane protrusion. But in studying filaments of the worm major sperm protein (MSP) using electron tomography, the authors noted that filaments became more loosely packed as they elongated.

“Polymer physicists have known for a long time that shorter rods pack more tightly,” says Roberts. By contrast, longer rods, regardless of composition, enclose more empty space. Measurements from the new images were consistent with this effect, and simulations confirmed that growing rods exhibited the same length-dependent space constraints as static ones. An MSP mutant that polymerized 2.4 times more slowly reduced the protrusion rate by 3.4-fold, indicating that both polymerization and packing-based expansion contribute to protrusion.

“It’s plausible that this effect may occur in actin systems as well,” says Roberts, although actin-based protrusion differs in its molecular details, including the use of cross-linking proteins.

Miao, L., et al. 2008. Proc. Natl. Acad. Sci. USA.

doi:10.1073/pnas.070841605. [Abstract]

Richard Robinson

rrobinson@nasw.org

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Journal : Embryos experience tension

Published online 7 April 2008
doi:10.1083/jcb.1812rr4
The Journal of Cell Biology
© The Rockefeller University Press, 0021-9525 $30.00

Embryos experience tension

Tension helps sort (top to bottom) ectoderm (red) and mesoderm (green) cells into ordered layers in vitro. Heisenberg/Macmillan

Tension helps organize tissue layers in early embryos, according to Michael Krieg, Carl-Philipp Heisenberg (Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany), and colleagues.

For Heisenberg, “The big question is what factors are important in cell sorting and tissue organization during development.” Differential adhesion among cells has been the most prominent hypothesis. Differences in cell cortex tension, which is produced by actomyosin contraction, have never been carefully measured before.

Using an atomic force microscopy probe, the authors measured the resistance to deformation in cells from each layer of the embryo. They found that tension was highest in the outermost layer, the ectoderm, followed by the endoderm and finally the mesoderm. By inhibiting myosin, the authors reduced ectoderm tension to match that of the other cell types. Ectoderm tension was also reduced when cells were treated with a growth factor that diverted them to a nonectodermal fate.

Cells sorted according to their tension rather than adhesion levels. When mixed in vitro, ectoderm cells partitioned to the inside, surrounded by meso- and endoderm cells. This inside-out arrangement is probably due to the absence of yolk cells and other in vivo factors that interact preferentially with each layer. Treatment with actomyosin inhibitors, which did not change adhesion, prevented ectoderm cells from burrowing inwards, indicating that cells rely on tension differences for sorting.

“In textbooks, you will read that differential intercellular adhesion of cells is sufficient to explain their sorting behavior,” says Heisenberg. “But it’s not just the adhesion that’s doing it. It’s a combination of several factors, including adhesion and tension.”

Krieg, M., et al. 2008. Nat. Cell Biol. doi:10.1038/ncb1705. [Abstract]

Richard Robinson

rrobinson@nasw.org

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Journal : Transcribing "silent" DNA resilences it

Published online 7 April 2008
doi:10.1083/jcb.1812rr5
The Journal of Cell Biology
© The Rockefeller University Press, 0021-9525 $30.00

Transcribing "silent" DNA resilences it

Entering mitosis, Swi6 (green) disengages from histones, allowing demethylation and transcription in S phase. RNAi remethylates the histones to complete the cycle. Kloc/CSHL

Transcription of heterochromatin during S phase is the key to the faithful inheritance of silenced DNA at the centromere, according to Anna Kloc, Rob Martienssen (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), and colleagues.

Histone methylation keeps heterochromatin condensed and the genes within silent. Yet the same methylation patterns are passed on to daughter cells. This inheritence requires RNA interference—and thus transcription. “That’s a paradox we’ve been looking at for several years,” says Martienssen. To resolve the paradox, the authors examined yeast centromeres for changes in RNA transcripts, histone modifications, and RNAi activity throughout the cell cycle.

As they enter G2, methylated histones at the centromere link to a major heterchromatin structural protein called Swi6, which in turn binds to cohesin, thereby tying the replicated chromosomes together. But during mitosis, histone phosphorylation knocks off Swi6; its removal is a prerequisite for transcription. The authors showed that this loss of Swi6 was accompanied by histone demethylation, which is also required for transcription. After passing through G1, the exposed centromeric sequences were transcribed during very early S phase. The transcripts were quickly processed by the RNAi machinery into siRNAs, which the authors had previously shown directs the methylation machinery back to the same demethylated histones at the transcribed centromeric sequences. So by the end of S phase, those sequences were remethylated and beginning to rebind Swi6 and cohesin, forming true heterochromatin again.

The timing makes sense. “S phase is exactly when you would need to modify histones to inherit epigenetic modifications,” says Martienssen, since that is when both copies can be targeted at once.

The authors also showed that temperature elevation inhibited the whole process. If the same is true in other systems, it might explain why some plants require a cold period before they can flower. This necessity depends on RNAi silencing and heterochromatin and requires cell division, suggesting that cold-driven gene modifications inherited during the winter trigger flowering in the spring.

Kloc, A., et al. 2008. Curr. Biol. doi:10.1016/j.cub.2008.03.016. [Abstract]

Richard Robinson

rrobinson@nasw.org

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Journal : Polarizing for furrows

Published online 14 April 2008
doi:10.1083/jcb.1812iti1
The Journal of Cell Biology
© The Rockefeller University Press, 0021-9525 $30.00

Polarizing for furrows

Monopolar HeLa cells fail to generate a single polarity axis for cytokinesis when actin filaments are disrupted.

Splitting mitotic cells in two is not the one-way signaling road it once seemed, based on evidence from Hu et al. The group identifies a positive feedback loop that creates a furrow at cytokinesis. The findings also throw a wrench in the well-accepted dogma of microtubule dynamic instability.

Hu and colleagues devised a monopolar HeLa system to determine how the cytokinetic furrow is created. Monopolar spindles are useful because they can be forced into cytokinesis synchronously, but the chromosomes don’t get in the way as they would in bipolar cells. In the round, monopolar HeLa cells, the spindle and its chromosomes were organized in a radially symmetric manner. Gradually, however, this symmetry waned, and microtubules and cortical furrow components began to polarize toward one side of the cell.

The de novo creation of this asymmetry implies that a positive feedback loop exists. Previous models proposed that a one-way signal is sent from spindle microtubules to the cortex to create a furrow. But the new results indicate that the cortex must talk back to microtubules, to stabilize them and further ensure polarization. Polarization required microtubules, Aurora B kinase activity, actin, myosin, and RhoA, which activates cortical contraction.

Because the monopolar spindles lacked a spindle midzone, where plus ends of microtubules from opposite poles overlap, their plus ends were easily identified. The group was stunned to note that these microtubule tips appeared nondynamic and terminated evenly at a distance from the growing, polarizing cortex. Their behavior contradicts popular dynamic instability models and suggests that plus ends may be capped in monopolar spindles and perhaps even at the midzone of bipolar spindles.

Within the gap between the plus ends and the expanding cortex, Aurora B colocalized with actin filaments. In bipolar spindles, Aurora B shifts from the kinetochore to microtubule plus ends. The new results suggest that it then travels along actin filaments to signal from the spindle to the cortex and possibly back again.

Hu, C.-K., et al. 2008. J. Cell Biol. doi:10.1083/jcb.200711105. [Abstract]

Nicole LeBrasseur

lebrasn@rockefeller.edu

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Journal : Phosphorylation for break repair

Published online 14 April 2008
doi:10.1083/jcb.1812iti2
The Journal of Cell Biology
© The Rockefeller University Press, 0021-9525 $30.00

Phosphorylation for break repair

The retention of MRN (red) at dsDNA breaks (green) is lost when MDC1 phosphorylation sites are mutated (right).

A constitutive, phosphate-based interaction between two repair proteins may help provide instant, well-coordinated repair of double-stranded DNA (dsDNA) breaks, according to two articles in this issue from Melander et al. and Spycher et al.

Nearly every step in dsDNA break repair requires MRN, from recognizing the break to activating signaling pathways to the mechanics of repair—or apoptosis, if repair fails. MRN can bind directly to DNA, but past experiments revealed that it also associates with the histones wrapped around damaged sequences, through an adaptor protein called MDC1.

Both groups now dissect this chromatin association. The findings reveal that the connection depends on a heavily phosphorylated domain within MDC1, which docks to a subunit of MRN called NBS1. In both articles, MRN was displaced from dsDNA breaks in cells containing mutant versions of MDC1 that lacked the phosphorylation sites.

The link between MRN and MDC1 did not depend on the presence of damaged DNA, however; even undamaged cells had phosphorylated MDC1. This phosphorylation depended at least in part on the constitutive and ubiquitous kinase, casein kinase 2 (CK2). Depletion of CK2 blocked the interaction between the repair proteins.

Repair aficionados often think that events taking place on the DNA itself are more important. But cells spend a great deal of energy coordinating the comings and goings of repair proteins on the chromatin, suggesting that these events might have evolved to improve repair precision. MDC1 is one of the earliest proteins to recognize histones at damage sites; by forming a constitutive link with MDC1, MRN ensures a rapid arrival to those same sites.

Previous results showed that MDC1 stays on damaged chromatin longer than MRN, indicating that the MDC1-MRN link is dynamic. This freedom may allow MRN to travel from the damage to the sundry other sites where it is needed, including the broken ends of the DNA.

The groups now hope to uncouple MRN and MDC1 without interfering with MDC1’s other binding partners by mutating only its CK2 target sites. They can then determine which of MRN’s sundry repair duties rely on its association with chromatin.

Melander, F., et al. 2008. J. Cell Biol. doi:10.1083/jcb.200708210. [Abstract]

Spycher, C., et al. 2008. J. Cell Biol. doi:10.1083/jcb.200709008. [Abstract]

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Jorurnal : Hauling tail

Published online 7 April 2008
doi:10.1083/jcb.1811iti4
The Journal of Cell Biology, Vol. 181, No. 1, 3-
© The Rockefeller University Press, 0021-9525 $30.00

Hauling tail

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During budding, glowing mitochondria remain stuck in the parent cell if Myo2's tail is mutated (bottom).

Solving a long-standing mystery, Altmann et al. have discovered that mitochondria get around a yeast cell in style. The organelles ride on the tail of a myosin molecule known as Myo2.

Mitochondria are always on the move. They change positions within the cell, come together to fuse, and split apart. During cell division or budding, they migrate into the daughter cell. The organelles scoot along actin filaments, but what powers them has stumped scientists. Prime candidates are myosin motors, which attach to actin and tow vacuoles and other organelles. But the evidence for the myosins' involvement in mitochondrial movement is mixed. Yeast strains with mutations in certain myosin genes show no abnormalities in mitochondrial structure or distribution. However, researchers have detected hesitant mitochondria in yeast with a mutant form of the myosin Myo2.

To clarify Myo2's contribution, Altmann et al. turned to yeast cells in which the level of Myo2 can be reduced by treating the cells with the antibiotic, doxycycline. The organelles normally link up to form a network. But in cells grown with the antibiotic, they clumped or curled into rings, suggesting disrupted movement. Altmann et al. found that isolated mitochondria lacking functional Myo2 cannot bind to actin filaments. The team then tested whether Myo2's tail, which carries vacuoles and other organelles, also hauls mitochondria. In yeast with mutations in the Myo2 tail, the researchers observed mitochondrial clumping. When a cell with one of these tail mutants sprouted a bud, few mitochondria moved in, Altmann et al. found. They conclude that mitochondria travel by boarding Myo2's tail. Up next: determining how Myo2 attaches to the mitochondrial surface.

Reference:

Altmann, K., et al. 2008. J. Cell Biol. 181:119–130.[Abstract/Free Full Text]

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Journal : Short proteins got no reason to leave

Published online 7 April 2008
doi:10.1083/jcb.1811iti5
The Journal of Cell Biology, Vol. 181, No. 1, 3-
© The Rockefeller University Press, 0021-9525 $30.00

Short proteins got no reason to leave

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The distributions of proteins with a long (red) and short (green) transmembrane domain don't overlap.

Acell uses a simple trick to keep certain proteins from leaving the ER: it bars them from the organelle's exits. Ronchi et al. show how cells determine which proteins to keep out.

Proteins due to be exported from the cell enter the ER, where they get bundled into vesicles that spirit them to the Golgi apparatus. One way that the ER prevents its resident proteins from departing by the same route is to exclude them from exit sites. But researchers didn't know what characteristics get a protein banished. Ronchi et al. suspected that the ER sorts proteins by their transmembrane domain (TMD).

To test the idea, the researchers altered human cells to manufacture two proteins that were identical except for their TMD, which was 22 amino acids long in one molecule and 17 in the other. The team had previously shown that the longer protein leaves the ER, while the shorter one stays behind. The new work shows that the 17–amino acid protein tended to avoid the ER exit sites. The longer protein, however, moved into and out of the sites, and a small amount of it accumulated there.

Where each protein ends up might depend on which lipids it associates with, the researchers speculate. Because its TMD is longer and more hydrophobic, the 22–amino acid version might seek stiffer lipid domains, which could gather at the exit sites. The shorter protein, by contrast, would fit in better with less orderly, thinner portions of the membrane.

Reference:

Ronchi, P., et al. 2008. J. Cell Biol. 181:105–118.[Abstract/Free Full Text]

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Journal : Stem cell slowdown

Published online 31 March 2008
doi:10.1083/jcb.1811iti1
The Journal of Cell Biology, Vol. 181, No. 1, 2-
© The Rockefeller University Press, 0021-9525 $30.00


Stem cell slowdown

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Slowly dividing follicle stem cells (green specks) are more abundant in mice lacking Zmpste24 (right).

Stem cells become sluggish in a disease that resembles premature aging, as Espada et al. show. The work is the first in vivo study to link the disease to stem cell abnormalities.

In some ways, children with Hutchinson-Gilford progeria syndrome (HGPS) resemble their grandparents. They lose their hair, their bones weaken, and they develop atherosclerosis, which usually kills them as teenagers. HGPS patients manufacture a defective version of lamin A, a key component of the nuclear lamina. Two years ago, Paola Scaffidi and Tom Misteli strengthened the link between aging and HGPS, showing that healthy people accumulate faulty lamin A at the edge of the nucleus as they age. One hypothesis suggests that mutant lamin A causes some infirmities of HGPS and aging by hampering stem cells.

Espada et al. tested this idea using a mouse model of HGPS. The animals fashion faulty lamin A because they lack an enzyme, Zmpste24, that helps trim the protein into its functional form. The team first measured the abundance of the skin's follicle stem cells, which promote wound healing and hair growth. To the researchers' surprise, the Zmpste24-deficient mice harbored more of these stem cells than did controls. However, these more numerous cells were reluctant to divide. In culture, stem cells from the mutant mice spawned smaller colonies than did cells from normal animals.

The researchers also found that abnormal lamin A disrupts the Wnt/β-catenin pathway that helps control the proliferation of stem cells. Zmpste24-lacking mice contained less of the active form of β-catenin and less cyclin D1, one of the division-promoting targets of the pathway.

Last month, Scaffidi and Misteli reported that mutant lamin A disrupted differentiation of mesenchymal stem cells (Scaffidi, P., and T. Misteli. Nat. Cell Biol. doi:10.1038/ncb1708). However, Espada et al. found that the follicle stem cells differentiated normally.

The mutant mice didn't show increased apoptosis by stem cells. But the suicide rate was higher among neighboring cells, whose signals nudge the stem cells to divide. The researchers conclude that abnormal lamin disrupts not just stem cells but also the surrounding cells that help control their behavior. The next step, the researchers say, is to look for similar defects in other stem cell types, such as hematopoietic stem cells.

Reference:

Espada, J., et al. 2008. J. Cell Biol. 181:27–35.[Abstract/Free Full Text]

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research : A yeast three-hybrid system that reconstitutes mammalian hypoxia inducible factor regulatory machinery

Research article
A yeast three-hybrid system that reconstitutes mammalian hypoxia inducible factor regulatory machinery
Maria L Alcaide-German, Alicia Vara-Vega, Luis F Garcia-Fernandez, Manuel O Landazuri, Luis del Peso
BMC Cell Biology 2008, 9:18 (10 April 2008)
[Abstract] [Provisional PDF]

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research : Regulation of DNA synthesis and the cell cycle in human prostate cancer cells and lymphocytes by ovine uterine serpin

Research article
Regulation of DNA synthesis and the cell cycle in human prostate cancer cells and lymphocytes by ovine uterine serpin
Maria B Padua, Peter J Hansen
BMC Cell Biology 2008, 9:5 (24 January 2008)
[Abstract] [Full text] [PDF] [PubMed] [Related articles]

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Research : The role of Drosophila Merlin in spermatogenesis

Research article
The role of Drosophila Merlin in spermatogenesis
Natalia V Dorogova, Elena M Akhmametyeva, Sergei A Kopyl, Natalia V Gubanova, Olga S Yudina, Leonid V Omelyanchuk, Long-Sheng Chang
BMC Cell Biology 2008, 9:1 (10 January 2008)
[Abstract] [Full text] [PDF] [PubMed] [Related articles]

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Journal : Structure of a new nervous system glycoprotein, Nr-CAM, and its relationship to subgroups of neural cell adhesion molecules

The Journal of Cell Biology, Vol 113, 1399-1412, Copyright © 1991 by The Rockefeller University Press

M Grumet, V Mauro, MP Burgoon, GM Edelman and BA Cunningham
Rockefeller University, New York, New York 10021.

We have identified and characterized a new glycoprotein in the chicken nervous system using immunological and molecular biological methods and we have examined its tissue distribution. Analysis revealed that this protein is very similar in structure to the chicken neuron-glia cell adhesion molecule, Ng-CAM, and to mouse L1. cDNA clones encompassing the entire coding sequence of this Ng-CAM related molecule, called Nr- CAM, have been isolated and sequenced. A glycoprotein containing one major component of Mr 145,000 on SDS-PAGE was purified from brain by lentil lectin affinity chromatography and FPLC, and its amino-terminal sequence was identical to that predicted from the Nr-CAM cDNA. The complete cDNA sequence encodes six Ig-like domains, five fibronectin type III repeats, a predicted transmembrane domain, and a short cytoplasmic domain. On Northern blots, nucleic acid probes for Nr-CAM recognized one major RNA species of approximately 7 kb and much lesser amounts of larger RNAs. Most of the same probes hybridized to single bands on genomic Southern blots, suggesting that Nr-CAM is encoded by a single gene that may be alternatively processed to yield several mRNAs. In support of this notion, two Nr-CAM cDNA clones had a 57-bp sequence located between the second and third Ig-like domains that was not found in two other Nr-CAM cDNA clones, and two other clones were isolated that lacked the 279-bp segment encoding the fifth fibronectin-like type III repeat. Antibodies against the purified protein and synthetic peptides in Nr-CAM both recognized a predominant Mr 145,000 species and a much less prevalent species of Mr 170,000 in neural tissues. Levels of Nr-CAM expression increased in the brain until approximately embryonic day (E) 12, followed by slightly lower levels of expression at E18 and after hatching. Immunofluorescent staining with anti-Nr-CAM antibodies showed that most neurons in the retina were positive at E7 and the pattern of expression became restricted to several layers on neuronal cell bodies and fibers during development. Anti-Nr-CAM antibodies labeled specifically cell surfaces on neurons in culture. Although the structure of Nr-CAM resembles that of chicken Ng-CAM and mouse L1, the identity with each of these neural CAMs does not exceed 40%. The differences indicate that Nr-CAM is distinct from Ng-CAM and L1, but there are sufficient similarities to suggest that all of these molecules are members of a subgroup of neural CAMs in the N-CAM superfamily.(ABSTRACT TRUNCATED AT 400 WORDS)

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