R E S E A R C H H I G H L I G H T S

Ca2+ is one of the most versatile sig-nalling factors that has been identi-fied so far, and the release of Ca2+

from the endoplasmic reticulum(ER) — which functions as an intra-cellular Ca2+ storehouse — has cru-cial roles in the regulation ofprocesses such as apoptosis and exo-cytosis. In Proceedings of the NationalAcademy of Sciences, Roger Tsien,John Reed and colleagues nowdescribe the development of animproved genetically encoded fluo-rescent sensor that can monitor Ca2+-concentration fluctuations in the ER([Ca2+]

ER), and they have used this

sensor to study the role of the anti-apoptotic protein B-cell lymphoma-2(Bcl2) in breast cancer cells.

Amy Palmer in the Tsien labora-tory started with the originalcameleon construct — two fluores-cent proteins (cyan fluorescent pro-tein (CFP) and citrine) separated by

calmodulin (CaM) and a CaM-bind-ing peptide. In the presence of Ca2+,CaM interacts with the CaM-bindingpeptide, and CFP emission decreasesas citrine emission increases, which isindicative of increased fluorescenceresonance energy transfer (FRET).Their aim was to change this cameleonto decrease its perturbation byendogenous CaM and to vary itsCa2+ affinity.

Using previously obtained struc-tural data, Palmer targeted salt-bridgeinteractions between CaM and theCaM-binding peptide. Compared tothe wild-type peptide, a mutant pep-tide with four charge reversals had a10,000-fold lower affinity for wild-type CaM. Palmer then made com-pensatory charge reversals in CaMwith the aim of restoring its affinityfor the mutant peptide. She namedthe mutant CaM and peptide pairDesign 1 (D1), and cloned it between

CFP and citrine to produce an alteredcameleon.

Next, Palmer studied the proper-ties of the D1 cameleon. In the pres-ence of Ca2+, the magnitude of theobserved FRET changes was compa-rable to that seen for the originalcameleon. In addition, the Ca2+ titra-tion curve for the D1 cameleonshowed that it is ideal for monitoring[Ca2+]

ER(that is, Ca2+ concentrations

in the low micromolar to hundreds ofmicromolar range). Furthermore, theD1 cameleon is not perturbed bylarge excesses of endogenous CaM,and it has a faster k

offthan previous

cameleons, so it can monitor rapidlychanging Ca2+ dynamics.

IN THE NEWS

Flies hooked on coffee? Well,Drosophila melanogaster does needcappuccino — for oocyte andembryonic polarity. It also requiresspire. Both genes contribute topolarity by affecting the actincytoskeleton. The cappuccino geneproduct, Capu, nucleates actin-filament formation through itsformin homology (FH) domains,but Quinlan et al. now report thatSpire (Spir) nucleates filaments bya totally new mechanism.

Spir contains four WASP-homology-2 (WH2) domains and astretch of acidic residues, whichindicated that it might activate theactin-nucleating Arp2/3 complex.But Spir induced the formation offilamentous actin clusters in theabsence of Arp2/3. This wasmediated by its N-terminal half,which contains the WH2 repeats

and the acidic domain, andoccurred by nucleating actinfilaments de novo with growth fromthe barbed end of the filament.

Next, the authors compared thenucleation activities andmechanisms of Spir, Capu andArp2/3. The FH1 and FH2 domainsof Capu and the N terminus of Spirnucleated actin polymerization atsimilar rates — slower than that ofArp2/3. And whereas the Arp2/3complex generated crosslinkedfilaments, filaments that wereformed by Spir and Capu weren’tcrosslinked. But, similar to Arp2/3,the N terminus of Spir capped thepointed ends of filaments.

Further investigation showedthat each WH2 domain and thesequences linking them havevarying roles in actin nucleation.Mutating all four WH2 domains

didn’t completely abolish nucle-ation activity, and Quinlan et al.found that linker-3 (L-3) showedweak nucleation activity, whichthey propose stabilizes two actinmonomers to promote actin-dimerformation.

The authors then tested theirhypothesis that, to form a newfilament, Spir binds several actinmonomers before assembling theminto a filament nucleus. Rod-likeSpir–actin complexes of four actinmonomers aligned along theirlength were seen, consistent withWH2 domains binding and align-ing actin monomers end to end.

Because the last two WH2domains connected by L-3 seemedto comprise the functional core ofSpir, the authors propose that thisregion mediates the formation ofan actin dimer — the main kinetichurdle to nucleation. A third andfourth monomer are then added bythe first two WH2 domains. Spirmight then stack monomers toform a nascent filament, and rapidfilament elongation from the stable

Inspired filaments

C Y TO S K E L E TO N

92 | FEBRUARY 2005 | VOLUME 6 www.nature.com/reviews/molcellbio

A changed cameleon

C A LC I U M

Loss of silencing leavespatients speechlessChildren with Rett syndromepresent autistic-likebehaviour and specificclinical signs, such as handwringing and loss of speech.Scientists previously showedthat this X-linked neuro-developmental disorder iscaused by mutations in agene on the X chromosome,which encodes methyl-CpG-binding protein-2 (MECP2).

As MECP2 regulates thetranscriptional activity of othergenes, a team led by TerumiKohwi-Shigematsu of theLawrence Berkeley NationalLaboratory, California, USA,searched for MECP2 targetgenes that might bedysregulated in mice with adefective MECP2 gene.Among them was DLX5, theexpression of which almostdoubled in MECP2-knockoutmice.

The maternally expressedDLX5 gene showed loss ofimprinting in lymphoblastoidcells from Rett-syndromepatients. The scientists foundthat, in mice, intact MECP2was required for specifyingrepressive histone methylationin the region that containedthe DLX5 gene, and fororganizing a transcriptionallysilent chromatin loop. InMECP2-null cells, such a loopwas missing.

“The findings are animportant piece in a very bigpuzzle”, according to AlanPercy of the University ofAlabama, Birmingham, USA(ScienceNow, 21 December2004).

In humans, DLX5 has animportant role in thesynthesis of γ-aminobutyricacid (GABA), which is aneurotransmitter that hasbeen linked to otherneurological disordersincluding epilepsy andParkinson’s disease. So theloss of imprinting mightchange the GABAergicneuron activity. “The nextquestion is whether crankingup the DLX5 gene results insome of the problems of Rettsyndrome”, says Kohwi-Shigematsu (ScienceNow,21 December 2004).

Arianne Heinrichs