In Brief

Secretion is polarized into the yeast bud so that the bud grows in preference to the mother cell. Pruyne et al. (page 1931) report that this actin-based process is directed by actin fibers that extend into the bud, and not by the actin patches that are clustered near the bud tip. Most perturbations to the actin system depolarize both patches and cables, but Pruyne et al. find that the combination of a temperature-sensitive tropomyosin 1 mutation and a tropomyosin 2 deletion can be used to selectively rid the cell of cables. Tropomyosin is found on, and stabilizes, actin cables but is not present in actin patches. Actin cables disappear just one minute after shifting the double mutant to a restrictive temperature; another minute later two other molecules are no longer concentrated at the bud tip. These proteins—Sec4, a secretory vesicle GTPase, and Myo2p, an unconventional myosin V implicated in targeted secretion—become diffuse several minutes before actin patches begin to delocalize. When the mutant is shifted back to the permissive temperature, tropomyosin-containing cables are reformed, and Sec4p and Myo2p localization are reestablished within one to two minutes. Repolarization of actin patches takes 15–20 min. The tropomyosin double mutant should help identify the yeast cell polarity marker. Of the proteins that are localized to the bud tip, only those that remain localized in the tropomyosin mutant, and that are necessary for regeneration of polarized cables, will remain as the leading candidates. This work and previous studies with Myo2p establish actin cable–based transport as the key event in yeast polarized secretion. Transport of mammalian melanosomes was also thought to be an actin-dependent process. But in mouse melanocytes, Wu et al. (page 1899) find that these pigmented organelles also undergo rapid, bidirectional, microtubule-dependent movements between the cell center and the periphery. A myosin V isoform encoded by the dilute locus is necessary for peripheral accumulation of melanosomes, but it functions primarily by capturing these organelles in the cell periphery. The striking difference between these two papers may reflect the different dimensions of the two cell types. Smaller yeast cells may preferentially use actin-based transport, whereas larger vertebrate cells use both shortrange actin-based transport and longer-range microtubule-based transport.

The Wiskott-Aldrich Syndrome protein (WASP) binds both Cdc42 and actin, and mutation of the human WASP gene leads to actin disorganization and defects in immune cell chemotaxis and antigen recognition. But it has proven difficult to define other members of the WASP pathway by biochemistry, as WASP overexpression results in a mess of aggregated filamentous actin. Bear et al. (page 1325) have tackled this problem with genetics. They started with Dictyostelium cells mutant in cAR2, one of four cyclic AMP receptors. These G protein-coupled receptors transduce chemotactic and morphogenetic signals. Loss of cAR2 function arrests the cells after they have aggregated but before the aggregate has formed a single tip, the precursor to the slug form. Deletion of the gene for SCAR (suppressor of cAR2) allowed cAR2 mutant cells to form a single tip; the same deletion caused each aggregate of otherwise wild-type cells to form multiple tips and multiple small slugs. Cells lacking SCAR had reduced filamentous actin but appeared to chemotax.
SCAR shares similarity with important WASP domains-including a characteristic acidic domain and domains that bind actin and profilin-but SCAR has a unique domain in place of WASP's Cdc42-binding site.
These are the first experimental data that link, albeit indirectly, a WASP-related protein with a seven-transmembrane receptor. The data also define a new gene family, distinct from WASP. A Dictyostelium WASP is already known, and Bear et al. identify SCAR homologues in humans, mice, and worms. Budding yeast, an organism that does not chemotax, has a WASP but no SCAR.

Erv14p Loss Causes a Specific Transport Defect
On page 1209, Powers and Barlowe report the discovery of Erv14p. This integral membrane protein is a component of COPII vesicles, which transport proteins from the ER to the Golgi. Erv14p is not needed for general transport; deletion mutants are viable. But the haploid mutants often fail to place their bud site in the normal axial position, next to the site of the previous cell division. The errors arise because Axl2p, an integral membrane protein that helps define the axial bud site, gets stuck in the ER.
A handful of other proteins have been linked to the transport of specific secretory cargo from the ER, so Erv14p's function is not unprecedented. But the next step has not been taken: no one has detected a physical interaction between a putative cargo receptor and its cargo. Such a link is predicted, given that secretory proteins can be concentrated into transport vesicles.
Erv14p is related to cornichon , a Drosophila protein needed for the posterior localization of the determinant gurken . But is there a fly Axl2p? If so, suggests Barlowe, cornichon could help transport the fly Axl2p to the oocyte posterior, where a delivery system for gurken could use it as a landmark.

Migration Mechanisms Double Up on Function Selectins That Signal
Movement of polymorphonuclear leukocytes (PMNs) from the blood to tissues starts with inflammatory mediators such as histamine, which induce the expression of various adhesion molecules on the endothelial cells (ECs) that line blood vessels. But there is more to transendothelial migration than adhesion. PMN migration is accompanied by a rise in intracellular calcium levels in ECs and is prevented by inhibition of this calcium transient.
Candidates for initiating this PMN-to-EC signaling event include the binding of the integrin CD11/CD18 to its EC partner intercellular cell adhesion molecule-1 (ICAM-1), and the homotypic binding of platelet/endothelial cell adhesion molecule-1 (PECAM-1). All these molecules are needed for transendothelial migration. But on page 1381, Lorenzon et al. find that it is the binding of PMN carbohydrates to their selectin partners that is necessary to generate the signal.
They find that monoclonal antibodies to vascular cell adhesion molecule-1 (VCAM-1) and E-and P-selectin induce the calcium transient in activated ECs; antibodies to ICAM-1 and PECAM-1 have no effect. PMN adherence induces the calcium transient, but not when the PMNs are pre-treated with antibodies to sialyl Lewis x (sLe x ), the counter-receptor for E-and P-selectins. Monocyte adhesion experiments give similar results, although interfering with VCAM-1 function also prevents the transient. Interfering with CD18 and PECAM-1 function has no effect on calcium transients in PMN or monocyte assays.
Treatments that induce the calcium transient also cause a rearrangement of EC actin from a circumferential ring to stress fibers. These stress fibers may help ECs retract from each other to aid PMN migration.

ZAP-70 in T Cell Migration
Activated T cells also migrate into tissues, notably in autoimmune diseases. T cell migration is dependent on the integrin LFA-1 (CD11a/CD18) on T cells.
Roos and colleagues (page 1371) use the Syk kinase inhibitor piceatannol and a dominant-negative ZAP-70 kinase to show that ZAP-70 is required for LFA-1-dependent T cell migration. This is true both in vitro (for cell migration through filters) and in vivo (for metastatic spread of hybridoma cells). ZAP-70 is known for its binding to the T cell receptor, an event that is essential for T cell activation, but in the hybridoma used by Roos, there is no T cell receptor.
Roos notes that ZAP-70-inhibiting drugs, designed to block immune responses, may have unexpected additional benefits. "If you have an autoimmune disease you already The Journal of Cell Biology, Volume 142, 1998 have activated T cells," he says. "But the drugs would still have an effect on autoimmune disorders because they would prevent migration of T cells into tissues."

How Life Deals with Excess Starting S in M
The early cell cycles of Xenopus embryos take just 30 min each, but the dimensions of the egg are large, meaning that chromosome segregation takes longer in the earliest cycles. LeMaitre et al. report that Xenopus keeps up with the cell cycle clock by starting DNA replication before chromosome segregation has been completed (see page 1159). By early telophase, when the division furrow is just starting to form and the spindle hasn't totally disappeared, replication is well underway, as evidenced by the incorporation of bromodeoxyuridine and the presence of replication protein A (RPA) and the processivity factor PCNA.
Xenopus does this by forming karyomeres. These structures had been characterized previously as single chromosomes encased in lipid. LeMaitre et al. find that karyomeres form in late anaphase, while chromosomes are still segregating, and before the chromosomes are close enough to each other to be enclosed in a single nuclear envelope (although eventually the karyomeres fuse to form a shortlived nuclear envelope). The karyomeres include nuclear lamins, which are essential for the initiation of DNA replication. Karyomere formation is probably made possible by the vast store of proteins and membrane vesicles in the egg. This supply runs out as embryos approach the midblastula transition.
The karyomeres make each chromosome an independent replication unit. Senior author Marcel Méchali says that the need for an enclosure-be it a nuclear envelope or a karyomere-fits with the licensing factor hypothesis. "Once you have formed the karyomere you are safe," he says. "No more licensing factor can get in, and you can start replication without worrying about getting rereplication."