IFT172 turns traffic around
Sure enough, she and coworkers found the algae version of the microtubule plus-end tracking protein EB1 out on flagellar tips. This localization was disturbed in a temperature-sensitive IFT mutant called fla11ts. These mutant flagella accumulated IFT particles at the tips, as though the transport machinery was unable to head back into the cell body.
Pedersen and colleagues have now found that FLA11 encodes an IFT complex protein called IFT172. They see that IFT172 and EB1 interact, but only when IFT172 is not bound to some of the other IFT proteins. Pedersen hypothesizes that IFT172 helps to link the IFT machinery to cytoplasmic dynein 1b/2. “At the tip,” she says, “when IFT172 encounters EB1, we think it binds to EB1, and somehow that promotes the reorganization of the IFT particle.” This reorganization may switch the particle from kinesin- to dynein-mediated transport. Since Pedersen still needs proof that EB1 is involved, she plans to use RNAi to get at its flagellar function. NL
Actin-powered endocytosis in real-time
The beauty of the system is that one can light up endocytic proteins and actin and watch an endocytic patch being born, invaginating, and moving off into the cytoplasm. Additionally, yeast endocytic mutants can be screened using this system for any spatial or temporal problems caused by a protein's absence.
In a previous study, the group localized an endocytic receptor and its cargo to actin cortical patches, thus providing the first direct evidence for the patches as sites of endocytosis. By hooking GFP variants to actin, Arp2/3 activators, actin binding protein-1, and endocytic adaptor proteins Sla1 and Sla2, the group was able to order events roughly as follows: endocytic and actin-nucleating proteins are recruited to the site, actin is nucleated as the vesicle begins to form, scission occurs, and then an actin-powered vesicle moves into the cytoplasm.
Now the group, led by Marko Kaksonen and Chris Toret, is characterizing known endocytic mutants, especially those with mammalian homologues, to compare yeast and mammalian endocytosis. One yeast mutant, for example, gives a yo-yo phenotype in which the vesicle starts to move off the membrane before snapping back suddenly, perhaps an indication of the mutated protein's role in scission. Another mutant erupts volcano-like plumes of actin from the membrane.
The analysis should answer questions about actin's role in vesicle invagination, scission, and transport. “Until you do things in real-time,” says Drubin, “you can't appreciate how intricate spatial and temporal regulation really are.” KP
Early sorting for membrane proteins
Soluble, cytoplasmic proteins can be localized by controlling the location of their mRNAs, and thus the ribosomes that translate them. Polarized secreted proteins are thought, in contrast, to achieve their deposition after sorting in the trans-Golgi, where they are directed into apical- or basolateral plasma membrane.
Secreted proteins, however, can also be localized via precise mRNA placement. Catherine Rabouille (University Medical Centre Utrecht, Netherlands) showed that, in fly oocytes, these proteins undergo a quick exit from the ER and local transport to the extracellular space through the local exocytic machinery.
Fly oocytes do not have the usual Golgi ribbon found in mammalian cells; rather, their Golgi is separated into 1000 individual stacks, each associated with an ER exit site (tER) to form a tER-Golgi unit. The new results show that only tER-Golgi units that are near the gurken mRNA, located at the dorsal/anterior corner, secrete the protein at the same corner. Changes in mRNA location thus changed where the protein was made and secreted.
The ER is one big lumen, so Gurken must be prevented from diffusing into distant tER-Golgi units. Rabouille's group found that this is achieved by rapid export from the ER. When this export was blocked, Gurken was seen throughout the ER. Next is to determine whether other polarized cells, such as neurons, use a similar strategy for locating polarized secreted proteins. NL
ATP is released from umbrella and other epithelial cells in response to the pressure of a filling bladder, although the mechanosensory mechanism that lets out ATP is not known. Apodaca finds that this released ATP is the trigger for exocytosis in umbrella cells. “We can take away ATP and block exocytosis in stretched cells,” he says, “or add ATP to stimulate exocytosis [without filling].”
ATP is sensed by ion channels in the umbrella cell plasma membrane. Umbrella cells from mutant mice lacking the P2X2 or P2X3 ATP-binding channels failed to undergo filling-induced exocytosis. Calcium was also needed, suggesting that ATP-bound receptors either channel calcium directly into the cell or let in other ions that then open calcium channels.
Nerves running to the bladder also have ATP receptors. ATP might thus serve two functions: to expand the umbrella cells, and to let the nerves know that the bladder is filling. NL
Actin herds DNA
Animal oocytes undergo an asymmetric division that sets the spindle near the surface of the animal pole. All chromosomes must therefore be pulled near the surface—some from the far side of the nucleus. But spindle microtubules are relatively short, due to their high dynamic instability. Lénárt and Ellenberg now find that microtubules are in fact too short to grab all the chromosomes in a starfish oocyte. “Over long distances,” says Ellenberg, “it's the actin cytoskeleton that's doing the movement.”
Actin seemed to form a meshwork with patches that attached to DNA. The net appeared at the time of nuclear envelope breakdown and then contracted toward the animal pole to bring the chromosomes within reach of spindle microtubules. Drugs that prevent actin polymerization caused chromosome losses, because the spindle captured only nearby DNA. Now, Ellenberg's group is looking for the polarity cues that control the direction of actin contraction. NL
Spindles adhere to adhesion pattern
Now, Manuel Thery and Michel Bornens (Institut Curie, Paris, France) have presented an elegant experiment to show that the adhesion pattern directs much more than just daughter cell shape—it may also align the mitotic spindle.
By plating thousands of HeLa cells on coverslips coated with a fibronectin “stamp,” the team could create cells in various shapes: rectangles, triangles, squares, and circles. After double-labeling cells for centrin and actin, the group used shape recognition software and video recording to measure automatically the orientation of division in relation to the long axis of actin cables.
It appeared that spindle orientation correlated with a cell's premitotic shape. But was this due to the overall cell shape or to the underlying adhesive pattern? Thery used a clever trick of “painting” fibronectin stamp patterns that would give similar shapes but with different adhesion patterns. For example, painted fibronectin in either a triangle or an “L” shape gives, in both cases, triangular cells, but with different adherence points and therefore different axes of actin cables.
By analyzing these types of manipulations of the adhesion pattern, the group shows that adhesion, not cell shape, determines the spindle orientation. The spindle is parallel to the premitotic actin–vinculin network and the plane of cell division is perpendicular to it. “We are proposing,” says Thery, “that it is really the internal forces of tension that drive spindle orientation,” possibly through adhesion-dependent signaling during interphase. KP
Young blood revives old stem cells
But were the stem cells losing their intrinsic ability to activate the Notch pathway, or was an extrinsic factor missing? Because satellite cells sit nestled in muscle tissue and respond to signals from nearby injured cells, the system is difficult to replicate in a cell culture or explant system.
In a collaborative effort, Conboy decided to use a parabiosis experiment—connecting the circulatory systems of a young mouse and an old mouse. “This made the system very clean,” says Conboy. “The whole mouse is still old, and the only contribution from the young mouse is blood.” In these pairs, an injury to the old mouse is repaired significantly better when young blood is supplied, and an in vitro test confirms that young blood serum restores Notch activation to old satellite cells.
Now Conboy has a head start on identifying the circulating molecules that change with age. And the parabiosis system, an experimental technique that has been around for hundreds of years, should prove useful for studying stem cell aging in other organs. KP
Traffic in the nanotube
Daniel Davis (Imperial College London, UK) presented observations that argue that perhaps both viewpoints are correct. Davis's studies of the immune synapse often have him asking whether processes happening between neural cells also occur between immune cells. And so it did not take him long, scanning live human macrophages and lymphocytes, to find nanotube structures forming between cells after they had “kissed” at the immune synapse.
The structures can be up to 150um, or 10 cell diameters, long and can persist for hours. The Davis lab's most striking observation, however, was that both large and small bulges can be seen traveling along the tubes from one cell toward another. Some bulges are large clumps of cellular material that deform the nanotube membrane as they travel; others are small presumptive vesicles that move within the diameter of the nanotube.
By measuring the direction and speed of these smaller vesicles, the group shows that the vesicle moves stepwise and at a constant speed when in motion. And it appears that the tubes support bidirectional movement. This suggests that, rather than simple diffusion, perhaps traffic is directed along cytoskeletal components. However, no such fibers have yet been identified in the tubes connecting immune cells.
Indeed, Davis knows that much hypothesis-testing remains to be done. Skeptics are quick to point to the fact that nanotubes form between pure liposomes. But Davis notes that, even if the tubes are simply a result of membrane biophysics, that still leaves room for cells to have evolved ways to exploit this property for cell.cell communication. KP
Putting signals in their place
The migration of border cells in the fly is controlled by two RTKs called PVR and EGFR. By sensing gradients of the PVF1 and Gurken ligands, respectively, the RTKs direct the border cells from somatic tissue to the oocyte. Rørth showed that overactivating or dampening RTK signaling had little effect on migration. Mutations that blocked RTK endocytosis, however, impaired migration.
In some systems, endocytosis brings receptors to an appropriate intracellular signaling compartment. But genetic experiments have shown that this is not the case for border cell guidance. Rørth finds that endocytosis affects the spatial extent, not the level, of signals downstream of the RTKs. “What is important here is not regulating receptor levels,” suggests Rørth. “It is ensuring that the signals stay localized. [For guidance,] cells need to measure where the signals are to find the front of the cell. They are not interested in what the levels of the signal are.”
Rørth hypothesizes that endocytosis brings active receptors to an RTK hot spot. The cell could thereby “exaggerate small differences in ligand concentration between its front and back, and make it into an all-or-none thing—in other words, polarize,” she says. NL
How to hug the curves
Antonny's group previously showed that ArfGAP1 is active only on membranes of high curvature. On budding COPI vesicles, ArfGAP1 helps Arf1 to hydrolyze its bound GTP, which in turn releases the COPI coat. ArfGAP1's ability to sense curvature is “a control in time and space for the budding vesicle,” says Antonny. “If the bud is young, it is flat, and the coat has not finished its job,” which is to induce more curvature. “As the buds get older, they are more curved, allowing access of the GAP.”
A yeast homologue of ArfGAP1, Gcs1, is also sensitive to membrane curvature. Its activity ranges several fold in magnitude based on the size of the liposome presented to it. Some GAPs of this family are not sensitive to membrane curvature, so Antonny's group used sequence comparisons to help them find the region that imparts the curve-sensing ability.
Gcs1 and ArfGAP1 share a short, central region, which the group found was necessary and sufficient for preferential binding to highly curved membranes. The stretch contains a tryptophan that is critical to binding. Antonny supposes that curvature introduces spaces between membrane lipids that are wide enough to fit the tryptophan and other bulky, hydrophobic side chains in the region. NL
MSP worms its way out of sperm
Eph receptors on the oocyte surface bind to MSP, which results in activation of a MAPK pathway that tells the oocyte to resume meiosis and ovulate. But how MSP gets out of the sperm and to the oocyte was rather a mystery. “Sperm lack ER, Golgi, and ribosomes [needed] for secreting proteins,” says Greenstein. “MSP has no recognizable signal sequence, and there's no evidence that it's proteolytically processed.” Instead, his lab is finding that MSP polymers may push their way out of the sperm.
Using high pressure–frozen samples for electron microscopy in collaboration with Kent McDonald (University of California, Berkeley, CA), the researchers spotted extracellular vesicles containing MSP sandwiched between two membranes (probably derived from the plasma membrane). Protrusions from which MSP escapes lacked a cofactor that is known to disassemble MSP filaments during motility.
The vesicles were labile, like time-release caplets of MSP. Immature sperm somehow made more stable vesicles, which makes sense for their time schedule—the young sperm conserve their MSP signaling and motility ability until the hermaphrodite matures. NL
Regulating sticky sisters
The anaphase promoting complex (APC) ubiquitin ligase, which regulates cell cycle progression, has two activators. Cdc20 turns on APC at the metaphase-to-anaphase transition so that it can degrade securin and promote sister chromatid separation. Then, after anaphase, Cdh1 activates APC to degrade Cdc20, setting up the cycle again for cohesion of the next sister chromatid pair. But for reasons unknown, Cdh1 stays active through late G1.
Susannah Rankin and Marc Kirschner (Harvard Medical School, Boston, MA) asked whether more Cdh1-dependent substrates of APC could be identified to elucidate other cell cycle mechanisms regulated by Cdh1.
Using frog egg extracts, which lack endogenous Cdh1, Rankin screened ∼60,000 in vitro expressed proteins for Cdh1-dependent degradation.
She pulled out an unknown protein that, on closer inspection, appears to regulate sister chromatid cohesion. When overexpressed, the protein, named sororin, leads to too much cohesion; when inhibited, the result is too little cohesion.
Rankin hypothesizes that sororin may be important for the establishment and/or maintenance of cohesion. If so, its degradation by Cdh1-activated APC during G1 would keep sororin levels low until after DNA synthesis, when chromatid cohesion would be established. If cohesion were around too soon, nonsister chromatids might be joined mistakenly. KP
On guard in the plant cell
By infecting plants with Pseudomonas syringae, Dangl's group has identified a complex of two R proteins, RPS2 and RPM1, and the protein they presumably monitor—the R protein interactor, RIN4. During infection, RIN4 can be modified by at least three bacterial proteins, including the cysteine protease AvrRpt2, which cleaves RIN4.
The Dangl lab now shows that the RIN4 cleavage product is detected by the plant to activate resistance, but is further manipulated by the pathogen so the pathogen can thrive in susceptible plants. Although this seems paradoxical, it actually fits the hypothesis—recognition of a modified host target alerts a resistant plant cell to the invader. A few R proteins can thereby recognize many different invaders who target the same cellular machinery.
Not surprisingly then, the RIN4 cleavage site domain is shared by other recognized proteins. In addition, all three bacterial effector proteins that target RIN4 are from different strains of P. syringae, showing convergence on the R protein complex.
As yet, it is unclear how RIN4 manipulations allow pathogens to survive in a susceptible plant. “Why does the cell have to guard RIN4?” asks Dangl. “It's guarded not once, but twice, and it is targeted [by pathogens] not once, but thrice.” KP
Unrest in the senescent cell
Senescence is triggered by a multitude of cell damage or stress signals, and although senescent cells can no longer divide, many of them become resistant to apoptosis. The number of these cells thus increases, and in fact they have been found at sites of aging-related pathologies.
Campisi's group had previously shown that senescent cells could provoke nearby cells that had already initiated the cancer process into a full-blown malignancy. Senescent cells “are not functioning as normal cells in normal tissue,” says Campisi. Now, the group has characterized the secretory profile of senescent human breast fibroblasts and found that the cells are pouring out chemokines, inflammatory cytokines, proteases, and growth factors. Why the cells do this is unknown, but one hypothesis is that they are locked in an activated wounding response.The team has also shown that senescent fibroblasts mixed in vitro with breast epithelial cells from a pregnant mouse alter the epithelial cells, lowering their milk production and increasing their formation of branching structures. So senescent cells influence both normal and precancerous cells in their environment.
Perhaps as an organism ages, the higher percentage of senescent cells synergizes with mutation accumulation to drive late-onset cancers. “A balance exists between tumor suppression and aging,” says Campisi. Early on, the tumor suppression mechanism predominates, but the later consequences of this may mean more tumors. “So the question,” says Campisi, “is can we uncouple them?” KP