In Brief

Fission yeast divide by constricting a myosin-rich medial ring and then septating. Defects in the septation initiation defective (Sid) group of proteins prevent both events. Sparks et al. (page 777) find that one of these proteins, Sid2p, is found at both the spindle pole body and the medial ring. Sid2p may carry a signal that anaphase is completed from the spindle pole to the medial ring, thus triggering cytokinesis.

Sid2p is at the spindle poles throughout the cell cycle, but appears at the medial ring only at the end of anaphase. Its appearance there depends on the formation of the medial ring, the presence of the medial ring protein Cdc15p, and an intact tubulin cytoskeleton. In cells that have neither microtubules nor a mitotic checkpoint, ∼10-fold less Sid2p reaches the medial ring, probably by diffusion.

In wild-type cells Sid2p may travel along cytoplasmic microtubules that reach from the spindle pole bodies to the medial ring. These microtubules have been shown to position the newly formed nuclei away from the site of division. “If these microtubules serve a dual function, positioning the nucleus and delivering Sid2p, then you're not going to get cytokinesis until you position the nucleus correctly,” says senior author Dannel McCollum.

Sparks et al. show that Sid2p is an active kinase that requires all other Sid proteins for activation. The other Sid proteins are found at the spindle pole body, and may be detecting the arrival of chromosomes at the pole at the end of anaphase. With the characterization of Sid2p, it is finally clear how these proteins influence events at the distant medial ring.

Correia et al. find that the actin cross-linker fimbrin can also bind the intermediate filament protein, vimentin (page 831). The interaction is transient, involves soluble not filamentous vimentin, and occurs only in adherent cells, at contact sites where the cell is actively spreading.

Vimentin is known to be delivered to the peripheral regions of certain cells by a microtubule-dependent mechanism; thus, fimbrin may come along for the ride. The interaction domains mapped by Correia et al. suggest that vimentin binding interferes with the actin-bundling of fimbrin, and fimbrin binding interferes with vimentin filament assembly. Once the complex arrives at focal contacts, however, it may fall apart to allow both actin bundling and vimentin filament assembly. Vimentin phosphorylation is one of the many possible mechanisms for switching association state, as phosphorylation is known to control vimentin filament assembly.

Replication protein A (RPA) and Mcm2 both load onto DNA in frog extracts before DNA replication commences, but they do not load onto the same sites. Do one or both of these proteins identify prereplication complexes? Dimitrova et al. find that, in Chinese hamster ovary cells, Mcm2 is recruited to chromatin at the end of mitosis, but RPA does not assemble on DNA until just before the initiation of DNA replication (page 709).

The early association of Mcm2 is consistent with its proposed role in licensing DNA replication, and Mcm2 staining is displaced as a particular section of DNA is replicated. In contrast the staining of RPA is more consistent with a role in the replication process itself. The prereplication RPA foci seen in earlier studies may reflect a process such as DNA repair.

Dimitrova et al. use a double labeling technique to differentially label early- and late-replicating DNA in one cell cycle, before following the DNA's fate in the following cell cycle. The timing of Mcm2 association does not vary between early- and late-replicating DNA, suggesting that some other factor determines when replication is turned on.

On page 801, Myster et al. provide the first full-length sequence of a flagellar inner arm dynein heavy chain. By transformation of a Chlamydomonas mutant, they show that only the amino-terminal 143 kD of this 522-kD protein is needed for assembly of the inner arm I1 complex, but that the rest of the motor is needed for phototaxis if the flagellar outer arm is missing.

The power of the flagellar wave comes from the proteins of the outer arms, including three dynein heavy chains. Outer arm mutants have a slower beat frequency and appear more jerky. The inner arms are closer to the control center, the central microtubule pair and radial spokes that apparently turn the dyneins on and off, and without the inner arms the flagella produce an aberrant waveform and slower cell movement.

Flagellar movement may be controlled by mechanical feedback and cycles of phosphorylation and dephosphorylation. But to pin this action on specific proteins, it will be essential to localize various proteins within the flagellar structure. For the inner arms this is no simple feat: there are seven different complexes containing at least eight different dynein heavy chains. Myster et al. make a start by isolating the gene encoding the 1α dynein heavy chain.

Transformation of various constructs of this gene into the 1α dynein heavy chain mutant results in rearrangement or fragmentation of the gene such that only the amino terminus is expressed. This is sufficient for assembly of the I1 complex, although the absence of the motor domain is visible as a missing lobe in the tri-lobed structure. The incomplete I1 complex restores phototaxis, but only in the presence of the outer arms. This suggests that outer and inner arms must cooperate to achieve wild-type motility and phototaxis.

Phosphoinositides and the enzymes that metabolize them are involved in various protein trafficking steps, and in mediating the action of endocytosed receptors. Now Gaidarov and Keen find that phosphoinositide binding by the endocytosis protein AP-2α is essential for the incorporation of this adapter protein into clathrin-coated pits and therefore for productive endocytosis (page 755).

Gaidarov and Keen use a triple, lysine to glutamic acid mutation to abolish phosphoinositide binding. The mutant polypeptide is incorporated normally into the heterotetrameric AP-2 complexes, but the protein no longer assembles into coated pits at the plasma membrane. Higher levels of the mutant protein displace endogenous AP-2 and disrupt endocytosis. The exact role of the phosphoinositide is not known, but a function in recruitment of AP-2 to the site of coated pit formation on the plasma membrane is likely. These workers recently reported that arrestins require phosphoinositides to recruit activated G-protein–coupled receptors to coated pits. Thus, inositides may have a general function in the initial steps of the endocytosis pathway.