1663mr Page 308 Thursday, July 22, 2004 1:10 PM

Meeting Report
In May, the Japan Society for Cell Biology held their annual meeting in Osaka. For some of us, this year’s meeting was aspecial treat; presentations were given in English for the first time in the meeting’s 57-year history. The conference organizers,including President Yoshimi Takai (Osaka University), did a superb job of attracting top cell biologists from not only Japan,
but also far overseas. Each symposium was an especially cohesive collection of presentations, and topics included microtubule targeting and function, tissue remodeling, membrane trafficking, ubiquitylation in DNA repair, noncoding
RNAs, structural analysis of transmembrane signaling, and advances in live-cell imaging. Poster sessions included
discussions in Japanese when appropriate, but English was also heard throughout. Attendees and speakers included
both established and young, up-and-coming scientists who enjoyed top-rate science in an international atmosphere.

PIP2 unlocks linkers

Kinesins deliver stabilizing goods

PIP2 binds to and activates the closed form of ERMs.

PIP2-mediated activation restricts ERM opening to the membrane.
Adhesion molecules can then lock the ERMs at adhesion sites.
The adhesion molecules may also recruit ERMs by pooling PIP2.
Hakoshima showed that adhesion proteins such as ICAM-2 containpositively charged regions just inside the cytoplasmic side of themembrane that remain accessible when ERMs bind. ICAM2 clustering
may therefore concentrate negatively charged PIP2 at adhesion sites,
bringing in more ERMs.
PIP2 production is enhanced by Rho, which also activates a candidate
ERM kinase. ERM phosphorylation stabilized the open structure,suggesting that high Rho activity may strengthen adhesions via multipleroutes. References: Hamada, K., et al. 2000. EMBO J. 19:4449–4462.
Hamada, K., et al. 2003. EMBO J. 22:502–514.

308 The Journal of Cell Biology | Volume 166, Number 3, 2004

Some kinesins destroy microtubules by chewing themup. But others have stabilizing effects that have notbeen explained. David Pellman (Harvard Medical School,Boston, MA) presented evidence that, in yeast, thesemotors can be stabilizing because they deliver plus-end
binding proteins to microtubule tips.
In budding yeast,
astral microtubules are
more stable duringmitosis than during interphase, which allows
them to grow toward
the bud site for correct
spindle positioning.
Pellman finds that thesechanges in microtubule
stability correlate withfluctuations in the levelsof the Kip2 kinesin.
Pellman was able to
track this motor and its
cargo on individual
microtubules. Kip2’s
Kip2 (red) takes Bik1
cargo included Bik1,(green) to the plus ends
a plus-end binding pro- of microtubules.
tein known to stabilize
microtubules. During mitosis, Kip2 levels were highest
and so Bik1 was carried out to the plus ends.
Kip2 was also needed to bring dynein, a minus-end–
directed motor needed to pull the spindle into the budat anaphase, to microtubule plus ends. From there,dynein was transferred to the cortex. A receptor on
the cortex, possibly Num1, seems to activate dynein.
“When [dynein] hits the cortex, we see showers of[dynein] speckles tracking toward the minus ends,”
says Pellman.
Kip2 levels are low until mitosis, so the plus ends ofinterphase microtubules have less dynein and Bik1.
Their absence seems to favor other plus-end binding
proteins, such as Kar9, which works with a myosin in G1
to guide spindle microtubules to the bud site. Pellman/Elsevier

Hakoshima

RM proteins link adhesion molecules to the cytoskeleton, but thislinkage must be restricted to the necessary place and time. Structural
analyses by Toshio Hakoshima (Nara Institute of Science and Technology, Nara, Japan) suggest that phospholipids take care of this regulation.
Inactive ERMs, such as radixin, are folded to mask both their actinand adhesion protein–binding (FERM) domains. This fold is lost in
vitro if either the actin-binding domain is phosphorylated or theFERM domain binds to PIP2. Both modifications occur in vivo, so
Hakoshima examined which was critical for radixin activation.
The analysis revealed that the phospholipid binding activates ERMs.
In the radixin crystal structure, the PIP2-binding site was accessible in
the folded form, whereas the phosphorylation site was buried within
the interface. Phospholipid binding to FERM opened the structure,thus exposing both actin- and adhesion molecule–binding sites.

Reference: Carvalho, P., et al. 2004. Dev. Cell. 6:815–829.

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The Journal of Cell Biology

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TEXT BY NICOLE LEBRASSEURLEBRASN@ROCKEFELLER.EDU

West Meets East: JSCB 2004
Osaka, Japan May 26–28, 2004

Silencing specificity

RSiomi/CSHL

NA-mediated silencing begins with two types of RNAs. Those completely complementary totheir target (siRNAs) lead to message degradation, whereas those less perfectly matched (miRNAs)
block translation of their target. Both RNAs are produced by and carry out their very different
functions within an RNAi-induced silencing complex (RISC). Until recently, all RISCs were considered equal. But evidence presented by Mikiko Siomi (University of Tokushima, Japan) suggeststhat RISCs are customized for the two RNAs with an appropriate Argonaute (AGO) family member.
Flies have multiple AGOs, but Siomi shows that at least AGO1 and AGO2 are not interchangeable.
AGO2 mutants were blocked in single-stranded siRNA production but could make miRNAs.
The siRNA duplex is normally made by Dicer-2 cleavage of a long dsRNA, and AGO2 was needed
RNAi inhibition of ftz (top) does
to unwind this siRNA duplex, although no helicase domains have been identified in AGO2.
not happen if AGO2 is missing
AGO1, in contrast, was needed for miRNA accumulation and formation of the miRNA-containing(bottom).
RISC, but was dispensible for siRNA function. AGO1 associated with Dicer-1, which cleaves themiRNA precursor, and with both unprocessed and mature forms of the miRNA. In the AGO1 mutant, there is much less mature miRNA,but its precursor did not accumulate in its place, so Siomi supposes that AGO1 stabilizes the mature miRNA after cleavage.
The separate AGO functions suggest that perhaps one RISC does not fit all. “I believe that the [RISCs] are distinguishable in termsof the protein components,” says Siomi, “because we do not detect AGO2 in the AGO1 complex, and vice versa.”
Making the RNAs is probably restricted to the complex with the appropriate AGO. The two complexes may talk to each other duringlater stages of silencing, however. “Perhaps at the second round or later . . . , the small RNAs are somehow exchangeable between
RISCs, since we can clearly detect mature miRNAs both in the AGO1- and AGO2-containing complexes,” says Siomi. “But this ismerely our speculation.” The biological significance of any such interchange remains to be determined. Microtubules: on the plus side
icrotubule plus ends hold the keys to their dynamics
and, thus, their targeting, as was presented by Yuko
Mimori-Kiyosue (KAN Research Institute, Kyoto, Japan).
Those keys are plus-end binding proteins such as CLASPand APC. Because the plus end is the primary site of growthand shortening, proteins that bind there are good candidates
for regulating directional growth. Mimori-Kiyosue has foundthat this is the case for at least two classes of plus-end binding
proteins, CLASPs and adenomatous polyposis coli (APC).
Based on their localizations, CLASP1 and CLASP2 arepoised to act as linkers, as they were found to bind to plus
ends and to the cortex independently. CLASP2 also boundto EB1, which sits specifically on the plus ends of growing
microtubule. CLASP knock-down experiments reduced thenumber of microtubules at the very edge of the cell, suggesting that CLASPs keep growing, EB1-bound microtubulesnear the periphery. Microtubules in cells with reduced
CLASPs also had shorter pause time and more depolymerization events. In the presence of CLASP, peripheral microtubules were stable yet dynamic, with more frequenttransitions from growth to shortening and from shortening
to growth than in the knock-down cells.
Mimori-Kiyosue’s experiments were done in nonmotile
HeLa cells, in which CLASPs were found at the edges oflamellipodia all around the cell. In migrating cells, CLASPs
localize to the leading edge, as has been shown in neuronal
growth cones and fibroblasts, and may therefore favor
microtubule growth in the direction of migration.

Mimori-Kiyosue

MPlus-end binding proteins such as APC (green)stabilize and orient microtubules (red).

Migrating epithelial cells also receive directional instruction from APC during wound migration. APC is transported
to plus ends by the KIF kinesin. Mimori-Kiyosue showedthat only when APC also binds to PDZ proteins, such as theDLG scaffolding protein, does directional migration result.
DLG localizes to the basolateral plasma membrane andmay thus help point stabilized microtubules in this direction.
Wounds that slice through a tissue will therefore be repaired
efficiently by sideways migration of cells. 
Reference: Mimori-Kiyosue, Y., and S. Tsukita. 2003. J. Biochem.
134:321–326.

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Reference: Okamura, K., et al. 2004. Genes Dev. doi:10.1101/gad.1210204.