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{{Movie header}} {| | width=360px|<mediaplayer width='320' height='280' image="http://cellbiology.med.unsw.edu.au/cellbiology/images/9/90/Bacteria_cell_division_2.jpg">File:Bacteria cell division 2.mp4</mediaplayer> | ===Bacteria cell division=== [[Bacteria_cell_division_2.jpg|100px|left]] :'''Links:''' [[Media:Bacteria_cell_division_2.jpg|MP4 version]] | [[Media:Namehere.mov|Quicktime version]] | [[Movies]] |} ===Reference=== <pubmed></pubmed>| [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC PMC] | ====Copyright==== {{JCB}} {{Movie footer}} [[Category:Movie]]

JCB Annotated Video Collection

This page is a linked summary of the full original JCB Annotated Video Collection

Papers have been placed into topic categories and subcategories. Within each subcategory, more recent papers are shown at the top of the list. The categorization of selected papers and videos is approximate, so it is worth scanning several sections for a desired topic. The same paper on microtubule function in neurons could, for example, easily find its place under either the microtubule or neurite-outgrowth heading.



Cell cycle proteins

Fzy and Fzr cooperate to destroy cyclin B in flies

Raff et al. examine two regulators of fly cyclin B destruction: Fizzy (Fzy)/Cdc20 and Fzy-related (Fzr)/Cdh1. Fzy/Cdc20 is concentrated at kinetochores and centrosomes early in mitosis, whereas Fzr/Cdh1 is concentrated at centrosomes throughout the cell cycle. In syncytial embryos, only Fzy/Cdc20 is present, and only the spindle-associated cyclin B is degraded at the end of mitosis. A mutant form of cyclin B that cannot be targeted for destruction by Fzy/Cdc20 is no longer degraded on spindles of syncytial embryos, but still targeted by Fzr/Cdh1 in cellularized embryos, albeit more slowly than normal. This suggest that Fzy/Cdc20 is responsible for catalyzing the first phase of cyclin B destruction that occurs on the mitotic spindle, whereas Fzr/Cdh1 is responsible for catalyzing the second phase of cyclin B destruction that occurs throughout the cell.

Chromosome dynamics

A motor helps holocentrics pull away from poles

Holocentric chromosomes in worms have kinetochores distributed along the length of the chromosome. Powers et al. show that the plus-end microtubule motor KLP-19 helps pull chromosomes away from a monopole. This may put tension on a mono-attached chromosome, swinging the attached kinetochore towards the pole that it is attached to and away from the opposite pole. Without this system a single kinetochore can attach to both poles leading to lagging chromosomes and errors relative to a normal mitosis.

Kinetochores switch to depolymerization at anaphase

Maddox et al. find that during anaphase both poleward flux of microtubules and net depolymerization of microtubules at the kinetochores contribute to the movement of chromosomes. During metaphase, however, flux is compensated for by net polymerization of microtubules at the kinetochores.

The polar wind in action

The distribution of chromokinesins along chromosome arms makes these motors excellent candidates for mediating the polar ejection force. This force, which projects chromosome arms away from the poles, has been proposed to aid in chromosome congression to the spindle midzone. Levesque and Compton test this model by injecting antibodies specific for the chromokinesin Kid.

They start by looking in cells that have monopolar spindles because of the injection of Eg5 antibodies. Polar ejection, which results in chromosomes spreading outwards from the spindle pole, can be seen in these cells. But in cells injected with antibodies to both Eg5 and Kid the chromosomes cluster around the single spindle pole.

In normal spindles the difference is less severe. Normal chromosome congression is perturbed only slightly by the projection of chromosome arms towards spindle poles, and only a minority of mitoses are delayed.

Merotelic attachments are a major source of segregation errors

A chromosome can be left behind during anaphase if it has a merotelic orientation, i.e., a single chromatid has its kinetochore attached to kinetochore microtubule bundles extending towards both spindle poles. Cimini et al.detect such merotelic orientations by light microscopy and confirm them by electron microscopy. They represent a major mechanism of aneuploidy not detected by the mitotic spindle checkpoint.

CENP-meta is a motor for maintaining chromosome alignment

Yucel et al. find that CENP-meta, a fly kinesin-like motor most similar to vertebrate CENP-E, is required for the maintenance of chromosomes at the metaphase plate (compare this video to that of a normal mitosis). Thus there are distinguishable requirements for either establishing or maintaining chromosome congression.

Spindle checkpoint

Dynein shuts down the spindle checkpoint

Injection of a prometaphase cell with dynamitin, to inhibit dynein, blocks a cell in metaphase. But this block is relieved by a second injection of anti-Mad2 antibodies, which inhibit the spindle checkpoint. Howell et al. propose that the mitotic block occurs because dynein's normal function is to transport several kinetochore proteins, including checkpoint proteins, away from the kinetochore and towards the spindle poles. This action of dynein helps shut down the checkpoint once kinetochores are correctly attached to the spindle.

Kinetochores may act as sites for catalytically sequestering checkpoint proteins

Howell et al. find that the spindle checkpoint protein Mad2 (red) is a transient component of unattached kinetochores, as predicted by the catalytic model. In this model, unattached kinetochores provide sites for assembling and releasing Mad2-Cdc20 complexes, which sequester Cdc20 and prevent it from activating the events necessary for mitotic exit.

Spindle construction

Microtubule ejection thins out spindles

Rusan and Wadsworth show that microtubules are ejected from centrosomes later in mitosis, thus thinning out the astral arrays in favor of the central spindle. This may help the central spindle to provide a unique signal specifying the position of the cytokinetic furrow.

Dynein takes depolymerization to the poles

Spindle microtubules constantly move towards spindle poles in a process called flux, but the length of the pre-anaphase spindle is maintained by microtubule polymerization at plus ends and microtubule depolymerization at minus ends near the poles. In theory the microtubules could be pulled towards the poles by depolymerization, but Gaetz and Kapoor separate the movement and depolymerization by inhibiting dynein action. The result is cessation of pole-localized depolymerization and thus elongating spindles; it appears that dynein normally transports the microtubule-depolymerizing motor Kif2a to the poles. But flux is not affected, suggesting that other translocating motors, not pole-localized depolymerization, drive flux.

Kinetochore-nucleated microtubules help build spindles

A new contributor to spindle assembly is described by Khodjakov et al. They find that microtubules can be nucleated from kinetochores of monooriented chromosomes, and those microtubules are then incorporated into spindles that are either recovering from a monopolar state or bipolar but recruiting a monooriented chromosome.

Interphase microtubules are dragged into the spindle region during prophase

The rapid disassembly of the interphase microtubule network during prophase was observed by Rusan et al. The microtubules move inward along other microtubules, sometimes even doing a U-turn before moving back toward the centrosome.

EB1-driven dynamics helps during spindle construction

Rogers et al. find that depletion of EB1, a protein that localizes to the plus ends of growing microtubules, suppresses the normal dynamic growth and shrinkage of microtubules, leaving only passive, lateral movements. In fly embryos, this results in the failure of spindle elongation seen during normal mitotic divisions.

Aurora-A is required for spindle assembly

Normal spindle assembly in worm embryos requires the activity of Aurora-A kinase, according to Hannak et al. They find that embryos lacking Aurora-A fail in the microtubule-independent accumulation of γ-tubulin and two other pericentriolar components, which results in a failure of spindle assembly in which asters collapse onto each other. In a later paper, the same authors showed that centrosomal arrays can still form in the absence of γ-tubulin, but γ-tubulin is necessary for formation of a full mitotic spindle, and is the kinetically dominant centrosomal microtubule nucleator.

Evidence for a possible spindle matrix

Kapoor and Mitchison investigate the possibility that a static spindle matrix helps tether the spindle. They find that the mitotic kinesin Eg5 remains relatively stationary even as the microtubule spindle fluxes towards the spindle poles. This could be explained if Eg5 is walking along microtubules at the rate of flux but in the opposite direction, but the authors find that Eg5 distribution remains stationary even when Eg5 motor activity is inhibited. This leads to the proposition that a spindle matrix may exist and have Eg5 as one component.

Skeletor as a candidate component of a spindle matrix

Walker et al. propose that Skeletor, a protein they identify from flies, could form the basis of a microtubule-independent spindle matrix. Such a matrix could stabilize force production by the microtubule-based spindle. The Skeletor-based structure can form independently of microtubules, and normally arises during prophase before the microtubule-based spindle has been completed (see also the enlarged image of a single nucleus, with microtubules in green and Skeletor in red). The two structures coalign during metaphase.

Daughter centrioles move and are then anchored

Piel et al. show that mother centrioles stay immobile during G1, but daughter centrioles [/content/vol149/issue2/images/data/317/F4/DC1/JCB9912094.V5.mov move] extensively in a process dependent on the presence of either actin or microtubules. The gradual decrease in these movements as the cell cycle progresses suggests that there is a maturation-dependent process of centriole anchoring.

Centriolar satellites are transported towards centrosomes

Kubo et al. find that particles called centriolar satellites (green) are [/content/vol147/issue5/images/data/969/F4/DC1/JCB9907121.V1.mov transported] to their final resting place near centrosomes.

Organelle partitioning

Mitochondrial replication factories

DNA replication factories in mitochondria divide even in the absence of mitochondrial DNA, according to Meeusen and Nunnari.

Myosin V may help partition the ER

Wöllert et al. find that, during mitosis, myosin V–driven movement of small globular vesicles along F-actin is strongly inhibited, but the movement of ER and ER network formation on F-actin is up-regulated. Thus F-actin may help partition the ER during cell division.

Peroxisomes segregate using Myo2p

Hoepfner et al. analyzed the movement of peroxisomes in budding yeast. In the absence of the dynamin-like protein Vps1p only one or two giant peroxisomes remained, but segregation still occurred. Peroxisome movement was abolished by latrunculin A treatment, and movement was also found to be dependent on the myosin motor Myo2p.

Golgi clusters partition during mitosis

Golgi partitioning during mitosis has been suggested to occur via fusion of the Golgi with the endoplasmic reticulum. In contrast, Jokitalo et al. find that the Golgi membranes split into small clusters that persist through mitosis and partition early in mitosis to two sides of the nucleus. These two sets of clusters are pushed apart during anaphase, and form the basis for the re-formation of the Golgi in the two daughter cells.

No tetraploidy checkpoint

Concentrated actin polymerization inhibitors can both induce cytokinesis failure and halt the cell cycle, leading some to propose the existence of a tetraploidy checkpoint that detects the aberrant binucleate state. But Uetake and Sluder show that lower levels of the inhibitors still result in cytokinesis failure but now allow continued division of the binucleate cells, suggesting that such a checkpoint does not exist in these cell types.

Minimal requirements for inducing cytokinesis

Cytokinesis is known to be induced by DNA-containing mitotic spindles, but Alsop and Zhang find that micromanipulated cells can still go through cytokinesis even if they have only an aster or bundle of microtubules.

Rho activation during cytokinesis

Rho-family GTPases are thought to stimulate contraction of the actomyosin ring during cytokinesis. Yoshizaki et al. find, based on FRET activity that rises when the GTPases are activated, that the dynamics of activation during cytokinesis are not quite such a simple story. Although RhoA is active at the cleavage furrow, the patterns for Rac1 and Cdc42 activity are not as clear.

Rings that promote cytokinesis

Tasto et al. characterize Mid2, a fission yeast homologue of the cell division protein anillin. Mid2 forms a ring around the center of dividing cells, colocalizing and splitting along with the septin ring. Both rings help promote late events in cytokinesis.

Centrosomes are needed for spindle orientation but not cytokinesis

Khodjakov and Rieder find that centrosomes are not necessary for a normal cytokinesis, but cells lacking centrosomes do suffer from a lack of astral microtubules and therefore sometimes misorient their spindles leading to failures in cytokinesis. Cells lacking centrosomes also fail to enter S phase in the following cell cycle.

Chloroplast FtsZ forms a ring at the division site

Bacterial FtsZ mediates cell division by forming a ring at the cell division site. Vitha et al. show that chloroplast FtsZ also forms a ring and thus probably acts in a similar manner.


Nuclear structure - Chromosomes

TopoII α is mobile

The DNA-decatenating protein topoisomerase II has been proposed as a stable scaffold for mitotic chromosomes. Although Tavormina et al. do not rule out this idea, they show that a fluorescent version of DNA topoisomerase II Œ± (topoII Œ±) protein turns over rapidly. This may allow topoIIα, which is concentrated toward the axes of mitotic chromosome arms, to quickly reach and relieve areas of chromosomal strain that develop during mitosis. In another study examining the localization of topoII, Christensen et al. come to similar conclusions.

The nuclear envelope may help retard origin firing

Time-lapse microscopy of GFP-marked origins allows Heun et al. to show that late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope during G1, at which time a modified chromatin structure may be established to retard origin firing.

Nuclear pores

Nuclear pore complexes are fixed in place

Daigle et al. report that nuclear pore complexes (NPCs) undergo limited movements that match the deformations of the nuclear envelope as tracked using a grid of bleached nuclear lamins. NPCs are therefore remarkably stable complexes, and are probably anchored to a protein network in the nuclear envelope.

Nucleoporins reassemble around post-mitotic chromatin

A conserved nuclear pore subcomplex was characterized and tracked by Belgareh et al., who found that the proteins were recruited during telophase in a rim pattern surrounding the chromosomes. A low level of staining was also apparent on the kinetochores throughout mitosis.

Nucleoli

Nucleolar re-formation after mitosis

Savino et al. follow the re-formation of nucleoli after mitosis. Prenucleolar bodies (PNB) form on the chromosome surface and nucleolar material flows along links between PNBs and towards a developing nucleolar organizer region (NOR). Eventually this leads to the fusion of nucleoli to form a single entity.

Processing complexes may help reassemble nucleoli

Nucleolar reassembly during telophase is shown by Dundr et al. to require mitotically preserved processing complexes.

Speckles

A splicing factor has limited mobility

Based on the limited mobility of a splicing factor, Kruhlak et al. determine that the factor undergoes frequent but transient interactions with relatively immobile nuclear binding sites, both when associated with speckles and when dispersed in the nucleoplasm. This a 3-D video that should be viewed using red/green 3-D glasses.

ER to and from Golgi

Sar1 makes tubular ER export sites

Sar1-GTP induces the formation of elongated tubules that are also seen during export from the ER in living cells. Aridor et al. therefore suggest that Sar1 links cargo selection with ER morphogenesis through the generation of these transitional tubular ER export sites.

Two distinct pathways from Golgi to ER: Rab6 and KDEL

Recycling of proteins from the Golgi to the ER via the KDEL receptor (red) is [/content/vol147/issue4/images/data/743/DC1/KDELR-CFP_YFP-Rab6_Color.mov distinct] from retrograde Golgi to ER transport dependent on Rab6 (green), as determined by White et al. Only the KDEL pathway is dependent on COP-I.

Endosomes

Endosome to trans-Golgi traffic is vesicular

Coexpression of fluorescent Rab9 and Rab7 by Barbero et al. revealed that these two late endosome Rabs occupy distinct domains within late endosome membranes. Rab9 is present on endosomes that display bidirectional microtubule-dependent motility, and Rab9-positive transport vesicles (rather than tubules) can be seen moving along microtubule tracks and fusing with the trans-Golgi network.

Recycling of endosomes by Arf6

Activation of nucleotide exchange on Arf6 causes an increase in both membrane internalization and the return of the resultant structures to the plasma membrane. However, when Brown et al. block Arf6 in its GTP-loaded ("on") state, this results in accumulation of endosome-derived vacuoles.

Actin tails form behind motile endosomes and lysosomes

Taunton et al. document the formation of actin comet tails (red) behind [/content/vol148/issue3/images/data/519/F1/DC1/JCB9908074.V1.mov motile endosomes and lysosomes]. The tails may help the organelles move around the cell, and influence the construction of the cytoskeleton.

Lysosomes

Lysosomes repair the plasma membrane

When cells are injured, calcium rushes in and prompts exocytosis of membranes to repair the gap. Jaiswal et al. confirm that the repairing membranes are from lysosomes.

Vam6p clusters and fuses lysosomes

Caplan et al. find that human Vam6p promotes clustering and then fusion of lysosomes.

Plasma membrane

A marker for sequential exocytosis

The SNARE protein SNAP25, say Takahashi et al., marks the plasma membrane after an initial exocytic event to allow rapid sequential exocytic events.

A myosin V moves yeast secretory vesicles

Secretory vesicles actively move to the site of exocytosis in yeast. Schott et al. find that multiple secretory vesicles often follow the same linear track and frequently enter and cross the bud. This movement requires the activity of the myosin-V heavy chain encoded by the MYO2 gene. When the predicted lever arm of this motor is progressively shortened (with the most extreme example being the 0IQ mutant), the vesicle movements are progressively slowed.

Rapid cycling of lipid rafts to and from the Golgi

Nichols et al. detect rapid cycling of lipid raft markers between the plasma membrane and the Golgi. Through selective photobleaching, they are able to study transport either out from the Golgi to the plasma membrane, or in from the plasma membrane to the Golgi.

Membrane docking at the immunological synapse requires Rab27a

Stinchcombe et al. find that normal membrane docking of lytic granules at the immunological synapse is defective in cells lacking Rab27a. In cells lacking other Rab proteins, polarization of the secretory granules is incomplete.

Visualizing the location and dynamics of exocytosis

Schmoranzer et al. use total internal reflection (TIR) fluorescence microscopy to visualize exocytosis in mammalian cells (e.g., see event on left side of video). The analysis reveals that there are no preferred sites for constitutive exocytosis in this system.

Toomre et al. use a combination of TIR microscopy (green, labeling molecules close to or at the membrane) and standard fluorescence microscopy (red, for molecules further from the membrane) to visualize [/content/vol149/issue1/images/data/33/DC1/Fig_1b.mov trafficking to and fusion with] the plasma membrane during exocytosis. Red dots turn yellow then green as they approach the membrane, and then explode in a burst of light as they fuse with the plasma membrane during exocytosis. The transport containers appear to be partially anchored at the membrane before fusion, and can undergo either partial or complete fusion events.

Caveolae

Caveolin helps traffic lipids

Pol et al. observe the formation of lipid droplets in cells expressing a dominant negative caveolin protein, and suggest that caveolin helps traffic lipids to and from lipid droplets.

Axonal

Special microtubules for getting into axons

Nakata and Hirokawa find that preferential transport of cargoes into axons is directed by a special population of microtubules, which have a high turnover rate and increased binding of the tip-binding protein EB1.

Retrograde transport in axons

Lalli and Schiavo measure retrograde transport in axons. They show that the transport vesicles carry both tetanus toxin and NGF, but do not colocalize with lysosomes.

Wound healing

Using acid to close a wound

The sodium-proton exchanger NHE1 is needed for normal migration during wound closing, according to Denker and Barber. In cells with a mutant NHE1 that cannot translocate ions, directed migration fails. Normal ion exchange by NHE1 may change the pH at the front of the migrating cells, but the direct consequence of such a putative change is not known.

ARNO induces migration

Healing of large wounds normally progresses with the leading edge of cells migrating smoothly as a unit. Santy and Casanova find that cells expressing ARNO pull away from the wound edge and exhibit a distinct fan-shaped leading edge and a trailing edge with a tail that often remains attached to the body of the monolayer. ARNO acts as an exchange factor for the small GTPase ARF6, leading to increased activation of both Rac1 and phospholipase D. These two independent pathways function together to increase cell migration.

Actomyosin flow, accumulation, and contraction heal a wound

Mandato and Bement find that wounds heal using two distinct components: a highly dynamic assembly zone, in which myosin 2 and actin preferentially accumulate, and a stable contractile actomyosin ring. The contractile nature of the apparatus is revealed in several experiments: corners of a wound round up; contacting fingers of actin pull in the edges of a wound; and breakage of the contractile ring prevents proper closure. Other videos reveal the flow of actin, whereas myosin accumulates with little obvious flow.

Dorsal closure resembles wound healing

During [/content/vol149/issue2/images/data/471/F1/DC1/JCB9910093.V1.mov fly development], the process of [/content/vol149/issue2/images/data/471/F3/DC1/JCB9910093.V2.mov dorsal closure] (see also another video) brings together epidermal cells to form a sealed tube. Kiehart et al. demonstrate that dorsal closure can be largely, but not fully, explained by a purse-string model in which the leading-edge epidermal cells contract to seal the epidermal layer. This suggests that fly dorsal closure has many similarities with mammalian wound healing.

Actin

Microtubule and actin movements are coordinated

Fluorescent speckle microscopy (FSM) allowed Salmon et al. to examine both microtubules (MTs) and filamentous actin (f-actin) in migrating newt cells. F-actin exhibited four zones of dynamic behavior: rapid retrograde flow in the lamellipodium, slow retrograde flow in the lamellum, anterograde flow in the cell body, and no movement in the convergence zone between the lamellum and cell body. MTs moved at the same trajectory and velocity as f-actin in the cell body and lamellum, but not in the lamellipodium or convergence zone. MTs grew along f-actin bundles, and quiescent MT ends moved in association with f-actin bundles. Thus f-actin movements have a profound effect on MTs in migrating cells, and MTs and f-actin may bind to one another in vivo.

Actin-dependent picket fences slow diffusion in the plasma membrane

Fujiwara et al. track the movement of single phospholipid molecules in the plasma membrane. Over the short-term, these molecules appear to stay within defined compartments, but after an average of 11ms they hop to an adjacent compartment. Over even longer time periods (an average of 0.33 s), the lipids hop between larger compartments. Similar compartments are not apparent on vesicles, as trajectories are less closely apposed. Trajectories within a single compartment are not significantly slowed relative to diffusion rates in vesicles, so the delays in hopping between compartments must explain the lower overall diffusion rate for lipids in cellular membranes.

The compartmentalization depends on the actin-based membrane skeleton, but not on the extracellular matrix, extracellular domains of membrane proteins, or cholesterol-enriched rafts. The authors propose that various transmembrane proteins anchored to the actin-based membrane skeleton meshwork act as rows of pickets that temporarily confine phospholipids.

An actin fragment that relaxes myofibroblasts

Myofibroblasts are specialized fibroblasts that can contract to aid in wound healing, possibly by using stress fibers containing α-smooth muscle actin (α-SMA). Hinz et al. join the N-terminal sequence of α-SMA to a fusion peptide. Application of the resulting protein to myofibroblasts relaxes the cells reversibly. Such an agent may be useful in treating fibrocontractive diseases.

A Rop GTPase controls pollen tube tip growth

Fu et al. show that Rop1At, a Rop GTPase belonging to the Rho family, controls actin dynamics and thus pollen tube tip growth in tobacco plants.

Myosin

Myosin recruitment drives the distribution of nuclei in fly embryos

As nuclei divide in the early fly embryo, they are actively distributed along the long axis of the embryo. Royou et al. show that the cortical contractions that drive this are accompanied by periodic accumulation of myosin to the cortex. Recruitment occurs at the end of telophase, correlated with the drop in cdc2/cyclin B activity, and results in a cortical contraction during interphase.

A sensor for the activity and abundance of MLCK

Chew et al. construct a sensor that can read out both the abundance (shown as peak height) and activity (red is inactive and blue is active) of myosin light chain kinase (MLCK), a protein that activates myosin during nonmuscle cell contraction. The sensor has a Ca2+/calmodulin binding site placed between two added fluorescent domains. When MLCK is activated by the binding of Ca2+/calmodulin, FRET between the two fluorescent domains is disrupted.

In contracting cells, MLCK is recruited to and activated along contracting stress fibers (also visible in a second cell). MLCK is also activated in the lamella of motile and stationary cells, and at the cleavage furrow during cytokinesis.

MLCK stimulates rapid contraction; Rho kinase stimulates sustained contraction

Katoh et al. report that the calcium-dependent myosin light chain kinase (MLCK) triggers rapid stress fiber contraction, whereas Rho-kinase elicits sustained contraction, which is necessary for maintaining stress fibers, focal adhesions, and cytoplasmic tension. The authors separate the effects of these two contractile systems by preparing contractile fibers either in glycerol (which maintains both contractile systems) or Triton X-100 (which removes the Rho-kinase system).

Microtubules

Tea1p rides on microtubule ends

Feierbach et al. confirm that Tea1p moves to the ends of fission yeast cells on microtubules. They find that Tea1p does so by attaching to the ends of microtubules, which deposit it directly at the cell surface. Tea1p is probably held on microtubule ends by Tip1p, as in cells lacking Tip1p the Tea1p wanders along microtubules.

Microtubule catastrophe under pressure

Janson et al. report that force stalls MT growth and induces rapid catastrophes visible as single and repeated events. Detailed measurements suggest the barrier acts simply by slowing tubulin addition, thus giving more time for structural changes leading to catastrophe. This behavior may make microtubules a more adaptable positioning device.

Dynactin and microtubules search out their organelle targets

Vaughan et al. visualize the p150Glued subunit of dynactin (a binding partner for cytoplasmic dynein). They find that it associates with growing microtubule plus-ends to form "comet tails." When these tails encounter Golgi-derived membranes, the membranes are seen to initiate rapid movement (see the same phenomenon in close-up). This supports the search-and-capture model in which microtubules probe the cytoplasm for organelles in need of transport. A similar mechanism may underlie microtubule-kinetochore interactions.

Tea1p travels to cell ends and keeps polarity factors anchored there

Tea1p is needed to keep fission yeast growing linearly; in its absence cells become bent and branched. Behrens and Nurse demonstrate that tea1p is transported on the plus ends of microtubules from the vicinity of the nucleus to the cell ends. Tea1p prevents the curling of microtubules around the cell ends, and helps retain polarity factors at the cell ends.

Cytoplasmic microtubules position the nucleus

Cytoplasmic microtubules in fission yeast run from one end of the cell to another. Tran et al. suggest that these microtubules position the nucleus by attaching to the nucleus and then pushing on the ends of the cell. They first show that the nucleus undergoes microtubule-dependent deformations, and then that these deformations correlate with growing microtubules pushing (see also a second video) against the ends of the cell.

Cortex-microtubule interactions position the nucleus and spindle

Adames and Cooper find that the budding yeast nucleus moves to the mother-bud neck via capture of microtubule ends at one cortical region at the incipient bud site or bud tip, followed by microtubule depolymerization. Subsequent spindle movement into the neck is mediated by microtubule sliding along the bud cortex, which can sometimes be seen to occur with free microtubules. In a later paper, Heil-Chapdelaine et al. found that the cortical protein Num1p provides an essential attachment point for the sliding machinery (see the color version, or grayscale version with full legend).

Kinesin

KIFC3 and dynein cooperate in Golgi positioning

Golgi membranes disperse in cells treated with nocodazole, but then recover their perinuclear distribution as microtubules regrow. In cells lacking the kinesin KIFC3, however, Xu et al. find that Golgi membranes remain scattered. Similar results are evident when the Golgi is initially dispersed by BFA treatment of either wild-type or kifC3-/- cells. The effect on kifC3-/- cells is only present when dynein is inhibited by cholesterol depletion or dynamitin expression, suggesting that dynein and KIFC3 cooperate in Golgi positioning.

Kinesin's neck linker drives processive movement

Conventional kinesin is a dimeric motor that moves along microtubules. When Tomishige and Vale restrained motion of kinesin's neck linker via an oxidative crosslink, the movement of kinesin was restricted to brief one-dimensional diffusion events. This indicates that conformational changes in the neck linker, not in the neck coiled-coil, drive processive movement by the kinesin motor.


Determining direction of movement

Rafts to the front and back

[%5Bhttp://jcb.rupress.org/cgi/content/abstract/164/5/759 Gómez-Moutón et al.] find that lipid rafts that include PI3 kinase localize to the front of moving cells; other rafts move to the back. Disruption of raft structure by extraction of cholesterol prevents both PI3 kinase localization and cell movement.

Cdc42 focuses the direction of movement

After inhibition of Cdc42 function by Srinivasan et al., cells fail to orient their movement correctly and instead form pseudopods in an irrelevant orientation.

Cells can move by either destroying or squeezing through the matrix

Wolf et al. find that cells can use either of two methods to move: they can create a path by proteolytically destroying extracellular matrix, or they can resort to amoeboid movement using propulsive squeezing through gaps in the extracellular matrix. Whereas proteolytic movement remodels the surrounding matrix via traction and bundling, during amoeboid movement the matrix is not affected. During amoeboid movement the cells are forced to squeeze through small holes in the matrix.

Electrotaxis uses a unique signal transduction pathway

Wound healing may be enhanced by the presence of electrical fields, which are induced by spatial and temporal variations in epithelial transport or electrical resistance. Similar fields have been shown to affect the migration of many cell types. As a model for such migration events, Zhao et al. examine the ability of Dictyostelium amoebae to undergo electrotaxis. They find that the early stages of signal reception and transduction are not shared between electrotaxis and the well-characterized, G-protein-dependent process of chemotaxis. Inositol phospholipids, which are concentrated at the leading edge of cells during chemotaxis, did not show an intracellular gradient during electrotaxis, and G-protein-coupled receptors were not redistributed. But the respective signaling strategies must converge somewhere upstream of directed actin polymerization, as an actin binding protein shows polarized localization during electrotaxis.

RhoA helps pick up tails of moving cells

Worthylake et al. look at monocyte movement and find that monocytes lacking RhoA activity have trouble picking up their tails.

Making and remodeling adhesions

Splitting podosomes to make new adhesive contacts

Podosomes are actin-rich adhesions in macrophages that have many similarities to the focal complexes used for leading-edge adhesion in other cell types. Evans et al. track the formation and turnover of podosomes at the leading edge of a moving macrophage. Fission and fusion events can both be seen; the fission may be explained by branching polymerization by actin. Both de novo assembly and fission events contribute to new podosome assembly at the advancing front of the cell.

Focal complexes behave differently at the front and back of the cell

Focal contacts at the front and rear of a migrating cell must behave differently so that cells can grab on at the front and let go at the back. Ballestrem et al. examine this behavior using a labeled integrin subunit. At the rear of the cell, RhoA induces high-density focal complexes that slide inwards. Integrin turnover is fast, possibly allowing polarized renewal and thus movement of the focal contacts. In contrast, the low-density focal complexes in lamellipodia induced by Rac1 are stationary and transient.

HA and CD44 work together to form adhesion-filled protrusions

Localized application of hyaluronic acid (HA), a major carbohydrate component of the extracellular matrix found mostly in skin, joints, and eyes, can promote the [/content/vol148/issue6/images/data/1159/F2/DC1/JCB0001041.V5.mov formation] of local adhesion-filled protrusions. As documented by Oliferenko et al., this is dependent on the CD44 adhesion receptor and the small GTP-binding protein Rac1. The resulting motility is used in processes from metastasis to wound healing.

Actions of microtubules

Arg connects microtubules and actin to help protrusion

Cells with Arg ruffle and protrude actively, with Arg concentrated in these protrusions. Miller et al. show evidence that Arg connects microtubules to actin bundles, thus allowing the microtubules to deliver protrusion-promoting proteins to the cell edges.

Microtubules target adhesions precisely

Krylshkina et al. demonstrate that microtubules probe close to the cell–substrate interface (microtubules close to the substrate appear darker) and specifically target cell–substrate adhesions. A suggestion of such targeting was seen by the same group in an earlier paper by Kaverina et al., and more examples of targeting can be seen here and here and here. The microtubules may be guided to the adhesion sites by actin bundles, and may bring molecules that regulate turnover of the adhesions.

Microtubules make a persistent push to the front

Wittmann et al. find that the few microtubules that make it into protrusions at the front of the cell grow more persistently than do other microtubules. Many more such “pioneer” microtubules are evident in cells with constitutively active Rac, even though Rac is normally thought of as a regulator of actin not microtubule dynamics. Pioneer microtubules are also swept backwards by actin retrograde flow.

Released microtubules dictate the direction of cell movement

Abal et al. find that short microtubules labeled at their growing ends with EB1-GFP track outwards towards the periphery. When the microtubules are instead anchored at the centrosome by overexpressed ninein, cells can still form polarized ruffles but they no longer move, suggesting that released microtubules help cells to move in a single direction.

Kinesin delivers a signal inhibiting adhesion sites

Microtubules target substrate adhesions and thus promote their disassembly. Krylyshkina et al. investigate which motor might deliver this signal. They first inhibit dynein, as evidenced by the dispersion of lysosomes, but this has no effect on adhesion site dynamics. Inhibition of kinesin, however, induced a dramatic increase in the size and reduction in number of substrate adhesions, mimicking the effect observed after microtubule disruption by nocodazole. Microtubules still target substrate adhesions. So conventional kinesin must be required only for the focal delivery of a component(s) that retards the growth or promotes the disassembly of adhesion sites.

APC is deposited from microtubules at the cell surface to promote outgrowth

Mimori-Kiyosue et al. report that the APC protein [/content/vol148/issue3/images/data/505/F6/DC1/JCB9907015.V4.mov moves along] and concentrates [/content/vol148/issue3/images/data/505/F7/DC1/JCB9907015.V5.mov at the ends] of microtubules. Shrinking microtubules [/content/vol148/issue3/images/data/505/F7/DC1/JCB9907015.V6.mov deposit] their load of APC near the [/content/vol148/issue3/images/data/505/F5/DC1/JCB9907015.V1.mov cell surface], where the APC (shown in green) may promote cell outgrowth or migration.

Microtubules deliver relaxing signals to contact sites

Kaverina et al. suggest that microtubules deliver localized doses of relaxing signals to contact sites to retard or reverse their development. Further, they propose that it is via this route that microtubules exert their well-established control on cell polarity. In spreading cells, for example, contacts turn over readily (arrow in left panel of [/content/vol146/issue5/images/data/1033/F1/DC1/JCB9904118.V1.mov video 1] but persist and enlarge when microtubules are depolymerized (arrow in right panel of [/content/vol146/issue5/images/data/1033/F1/DC1/JCB9904118.V1.mov video 1]). Decreases in contact size occur thanks to [/content/vol146/issue5/images/data/1033/F4/DC1/JCB9904118.V5.mov direct targeting] of microtubules to these sites, a phenomenon that can also be seen when microtubules [/content/vol146/issue5/images/data/1033/F6/DC1/JCB9904118.V8.mov regrow] after nocodazole treatment. The microtubules may deliver factors that inhibit contractility. Applying [/content/vol146/issue5/images/data/1033/F8/DC1/JCB9904118.V10.mov inhibitors] of contractility causes shrinkage of the contacts, loss of microtubules, and retraction of the cell.

Leukocyte transmigration

A docking structure for transmigrating leukocytes

During inflammation, leukocytes must exit the blood and enter the tissues via a process called transmigration. Barreiro et al. examine this process and the importance of ezrin, radixin, and moesin (ERM), which connect membrane adhesion receptors to the actin-based cytoskeleton. Moesin is clustered around a lymphoblast during both adhesion to the endothelium and transmigration across it, whereas VCAM-1 is clustered only during apical adherence, and I-CAM-1 is clustered during transmigration. These proteins provide the basis for an endothelial docking structure that plays a key role in the firm adhesion of leukocytes to the endothelium during inflammation.

Tether durations needed for leukocyte attachment and rolling

When a tissue is inflamed, leukocytes use L-selectin to attach to endothelial cells lining the blood vessels. The initial capture is followed by leukocyte rolling along the endothelium. Dwir et al. find that individual L-selectin tethers must persist for at least 20 ms to support continued rolling adhesion. The probability of successive tether formation apparently collapses when tether duration drops below this critical period. Failure in tethering can be seen, for example, when the tail domain of L-selectin is truncated by the 358stop mutation, which abolishes L-selectin's attachment to α-actinin and possibly other cytoskeletal proteins.

NCAM traps machinery at new synapses

For a contact between axon and dendrite to turn into a synapse, a great deal of machinery must be recruited. Sytnyk et al. find evidence that the neural cell adhesion molecule (NCAM) does this recruiting by anchoring intracellular organelles in nascent synapses. Clusters of NCAM, linked via spectrin to TGN organelles, translocate along neurites until the complex is immobilized via contact with NCAM in another neurite.

Actin and microtubule movements are coupled in growth cones

Actin and microtubules interact in growth cones, according to Schaefer et al. They make growth cone movies of both microtubules (MTs), and actin (with the first actin movie and second actin movie showing entire growth cones, and the third actin movie showing a close-up of the peripheral domain and transition zone). MTs enter the periphery by polymerization and are cleared by a combination of catastrophe and coupling to actin retrograde flow. The combination of MT polymerization and retrograde flow can also lead to treadmilling. Single MTs in the peripheral domain align predominantly along, or very near, filopodial F-actin bundles (also visible in close-up, and in another example that includes both tracking on actin and a catastrophe event). Retrograde flow can also lead to frequent MT bending, buckling, and breakage in the transition zone. Finally, actin arcs help bundle MTs into the central domain (see also a dual color movie). Schaefer et al. propose that the steady-state movement of F-actin and microtubules in the filopodia allows the system to adapt quickly: a slight decrease in retrograde F-actin flow, for example, would drive rapid microtubule advance along the filopodia.

Growth cones grab sites that can withstand tension

Like a rock climber, a neuronal growth cone senses its substrate to identify the route that will provide the best grip. Suter and Forscher use beads coated with the Aplysia growth cone adhesion molecule apCAM to show that the growth cone steers across the surface of a bead, but only if the bead is physically restrained. The tension from apCAM binding to the restrained bead leads to Src family tyrosine kinase activation, which then promotes the strengthening of apCAM-actin linkages. The stronger linkages further increase tension, until the apCAM-actin linkage is strong enough to guide growth cone extension. This process should drive growth cone migration along the path providing the best molecular grip.

PKC increases neurite growth by increasing microtubule growth

Normal microtubule dynamics at a neuronal growth cone are changed after activation of protein kinase C (PKC), as demonstrated by Kabir et al. PKC activation increases neurite outgrowth by increasing the lifetime of microtubule growth episodes two-fold, increasing rescue frequencies 1.7-fold, and decreasing catastrophe frequencies two-fold.

TI-VAMP is needed for neurite outgrowth

Martinez-Arca et al. are able to visualize vesicles containing tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) and show that this protein, probably via its fusion activity, is needed for neurite outgrowth.

Adhesion is necessary for elongation

The elongation of the worm body during development involves a concerted contraction of the epidermis, so when Petitt et al. mutated the cadherin-catenin system that helps hold cells together the result was a failure in elongation.

An adhesion protein for slime molds

Fey et al. identify SadA, the first adhesion protein known in the social slime mold Dictyostelium. Cells that lack the protein have problems – they move more erratically and sometimes their division is delayed or aberrant compared to wildtype cells. But they still stream normally after being starved (wildtype streaming is shown here).

Intermediate filaments help cells to hold together

Cells lacking the attachment between cytoplasmic intermediate filaments and the plasma membrane can be pulled apart more readily than can control cells. Huen et al. therefore conclude that intermediate filaments contribute to the strength of intercellular attachments.


Myo-endothelial progenitors isolated from muscle

Tamaki et al. examine cells from the interstitial spaces of murine skeletal muscle, and isolate a population of these cells that can form both endothelial and myogenic cells. The latter fate is clearly evident in vitro, as the progenitors give rise to cells that can undergo spontaneous contractions. This is seen in both round cells and myotubes. The myo-endothelial progenitors may contribute to postnatal skeletal muscle growth.

Skeletal muscle cells can couple with heart muscle cells

Heinecke et al. find that skeletal muscle cells (elongated myotubes) can become electrically coupled to heart muscle cells, which results in synchronous beating directed by the heart muscle cells. This suggests that skeletal muscle cells might become productively synchronized with heart muscle after being used to repair tissue damaged by a heart attack.

Release of platelets from megakaryocytes

Megakaryocytes [/content/vol147/issue6/images/data/1299/F1/DC1/JCB9909028.V1.mov release] platelets from long tube-like extensions called pro-platelets. Italiano et al. show that platelets are liberated from the ends of proplatelets, but that the number of ends is also increased by [/content/vol147/issue6/images/data/1299/F2/DC1/JCB9909028.V3.mov bending and branching].

Frog Dishevelled is transported dorsally to specify dorsal fate

Frog embryos specify their dorsal axis during the first cell cycle by transporting a determinant along microtubules from the vegetal pole to the prospective dorsal side. Miller et al. demonstrate that Dishevelled (Dsh) fulfills the criteria for such a determinant. The directional transport of vesicle-like organelles coated with GFP-marked Dsh can be [/content/vol146/issue2/images/data/427/F3/DC1/JCB9901105.V1.mov visualized] but is [/content/vol146/issue2/images/data/427/F5/DC1/JCB9901105.V2.mov disrupted] by treatments that disrupt dorsal specification. Once at the dorsal side, Dsh activates the Wnt signaling pathway to specify dorsal cell fates.


Execution proteases

Caspase cleavage of GRASP65 helps fragment the Golgi during apoptosis

Lane et al. find that during apoptosis the Golgi ribbon is fragmented into dispersed clusters of tubulo-vesicular membranes. Fragmentation is caspase dependent and GRASP65 (Golgi reassembly and stacking protein of 65 kD) is a caspase-3 substrate. Expression of a caspase-resistant form of GRASP65 partially preserved cisternal stacking and inhibited breakdown of the Golgi ribbon in apoptotic cells.

Cathepsin B can execute tumor cells

Apoptosis in a cancer cell line is shown by Foghsgaard et al. to be minimally dependent on proteases called caspases -- the usual executioners in apoptosis. Instead, a lysosomal protease called cathepsin B takes on this execution function, perhaps after being translocated to the cytoplasm. Many tumor cells upregulate cathepsin B because of its invasion-promoting properties, but this may come at a cost for the tumor cells, as the excess cathepsin B apparently makes the cells more sensitive to suicidal cell death.

Mitochondria

Apoptotic cells show a transient loss in mitochondrial membrane potential

Waterhouse et al. suggest that the transient loss of mitochondrial membrane potential that can be seen in individual apoptotic cells may be masked by cell-to-cell asynchrony when looking at a population of cells.

Ubiquitination

Misfolded proteins are actively transported into aggresomes

Misfolded proteins are gathered into structures called aggresomes, and García-Mata et al. find that the accumulation occurs via [/content/vol146/issue6/images/data/1239/F8/DC1/JCB9904099.V1.mov directed transport] to the aggresome along microtubules.

Autophagy

Apg5 helps form autophagosomes

Autophagosomes form as cup-shaped organelles that engulf large parts of the cytoplasm. As shown by Mizushima et al., Apg5, part of a ubiquitin-like conjugation system, localizes to the forming autophagosomes, and is essential for their formation.

Stress granules

Stress granule assembly is dynamic

Stressed cells inhibit translation initiation, and the resultant free mRNAs accumulate in stress granules (SGs). These can disassemble to yield polysomes once the stressor (in this case, arsenite) is removed. Kedersha et al. find that labeled SG-associated proteins rapidly and continuously shuttle in and out of SGs, indicating that the assembly of SGs is a highly dynamic process. Thus mammalian SGs may be sites at which untranslated mRNAs are sorted and processed for either reinitiation, degradation, or packaging into stable nonpolysomal mRNP complexes.

Mitochondria slow down for calcium

Mitochondria move along microtubules, but Yi et al. find that they slow down when calcium levels rise, probably so that they can help buffer the ion back to normal levels.

Activated Ras defines the front of chemotaxing cells

Sasaki et al. show that localized Ras activation at the front of slime mold cells allows successful chemotaxis to a point source.

TCR signaling without a synapse

Bunnell et al. dispute the idea that signaling through the T cell antigen receptor is dependent on assembly of a large, complicated structure called the immune synapse. They find that the signaling component ZAP-70 is rapidly recruited into small contacts that can almost immediately initiate calcium influxes. The ZAP-70 is initially but not eventually subject to rapid exchange.

Stress induces oscillations

Msn2 and Msn4 are two yeast proteins that move into the nucleus in response to severe stress; once there they help the cell to respond to stress. Jacquet et al. find that under intermediate stress conditions the two proteins instead oscillate into and out of the nucleus. Perhaps this oscillation prevents unnecessarily intense activation but keeps the cell primed for a full response should conditions worsen.


Dynein delivers HIV to the nucleus

McDonald et al. find that HIV uses dynein-dependent movement on microtubules, possibly to deliver viral genomes to the nucleus.

Listeria use cellular engulfment machinery to invade neighboring cells

Listeria monocytogenes bacteria [/content/vol146/issue6/images/data/1239/F8/DC1/JCB9904099.V1.mov move] around and between cells by co-opting the cell's actin polymerization machinery. Robbins et al. suggest that the bacteria exploit the cells' normal mechanism for engulfing neighboring cell surface fragments to achieve spread between cells. When the bacterial cells encounter a junction between two mammalian cells, they can be [/content/vol146/issue6/images/data/1333/F3/DC1/JCB9906163.V2.mov seen] either protruding (red P) into the neighboring cell or ricocheting (yellow R) off of it.


A dynamic Golgi is built from the ER

Brefeldin A is known to inhibit ER to Golgi transport, and thus to lead to Golgi disappearance through the continuation of Golgi to ER transport. Ward et al. use this treatment to observe the displacement of a variety of Golgi proteins, including those thought to be stable Golgi components. This furthers their view of the Golgi as a dynamic, rather than stable structure. The abrupt appearance of some of the proteins in punctate structures suggests that they do not track out from the Golgi, but are reassembled at ER exit sites. Ward et al. suggest that these exit sites are the building blocks for the Golgi apparatus.


Parallel fibropositors make for strong tendons

Canty et al. map out long, thin plasma-membrane extensions that they call fibropositors. These structures extend between adjacent cells and deposit collagen fibers in a parallel arrangement that confers strength to tendons.

Calcium inhibition of AC6 allows formation of gaps between endothelial cells

The barrier between blood and tissues, formed by endothelial cells, can be breached by inflammatory mediators. They elevate cytosolic calcium ([Ca2+]i) in the endothelial cells, thus increasing actomyosin tension and decreasing adhesions. This results in the generation of focal intercellular gaps that form a paracellular pathway to promote fluid, solute, and protein permeability. These changes are only possible, say Cioffi et al., because the [Ca2+]i also inhibits type 6 adenyl cyclase (AC6) in endothelial cells. When the authors boost cAMP levels by introducing the calcium-stimulated AC8 this prevents gap formation.

Matrix meshes can be strapped together with small-scale forces

Fibroblast explants in collagen gels, like cells in tissues, can mold the surrounding matrix. They exert mechanical forces that lead to the formation of ligament-like straps between the explants. Somehow, the micrometer-scale cellular traction forces produced by the explants end up generating millimeter-scale structural changes in the matrix. Sawhney and Howard studied this process with fiduciary beads and a computer algorithm. They found that the collagen forms a mesh of interconnected fibers. Small movements along one axis of this collagen mesh (the axis running between explants) generate a large movement perpendicular to the axis, which draws collagen into the strap.