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172 trends in CELL BIOLOGY (Vol. 9) May 1999

Baron et al. have developed a regu-latory system for the study of genefunction in higher eukaryotes thatallows the reversible control of theactivity of two genes, or two alleles ofone gene, in a mutually exclusiveway. It also allows the abrogation ofthe activities of both genes or bothalleles, thus permitting a null pheno-type. The system is based on twocomplementary versions of the tetra-cycline (Tc)-controlled transcription-activation system, whereby bindingof one transactivator (tTA) to its corresponding promoter preventsactivation in the presence of a tetra-cycline, whereas the other trans-activator (rtTA) requires tetracyclinefor activation of transcription. Thetransactivators are fusion proteinsconsisting of the Tet repressor, withrtTA containing four amino acid

substitutions in the Tet protein thatgive rise to the reverse phenotype,and the C-terminal portion of VP16from Herpes simplex virus. By varyingthe concentration of a single effectorsubstance, a tetracycline, the activityof two genes can be controlled in amutually exclusive way.

By mutating three amino acids ineach transactivator and a single base inthe operators, Baron and colleaguescreated transactivators that discrimi-nated in excess of 1000-fold more effi-ciently between the two new cognateoperators1. Additionally, as the trans-activators function optimally byhomodimerization, the dimerizationproperties of both transactivator pro-teins were altered so that heterodimerswere no longer produced. The trans-activators were further engineered byreducing the VP16 activation domains,

thereby minimizing possible sideeffects of high intracellular concen-trations of VP16 fusion proteins.

Transient- and stable-transfectionexperiments with the engineeredtransactivators demonstrated thatthe two genes could be regulatedseparately and quantitatively. One of many attractive experimental scenarios would be the generation of conditional mutants at the level oftransgenic organisms.

Total gene control

1 Baron, U., Schnappinger, D., Helbl, V.,Gossen, M., Hillen, W. and Bujard, H.(1999) Generation of conditionalmutants in higher eukaryotes byswitching between the expression of two genes, Proc. Natl. Acad. Sci. U. S. A. 96, 1013–1018

Many cells – from yeast to neurons –show some degree of asymmetry orfunctional polarization. Nowhere isthis better understood than in epi-thelial cells where tight junctions limitdiffusion between the apical andbasolateral membrane domains atsites of cell–cell contact. But, howabout neurons, which do not containany obvious physical barrier to sep-arate the axonal from the somato-dendritic plasma membrane?

Winckler et al. have now character-ized the presumptive diffusion barrierin hippocampal neurons in cultureusing immunofluorescence micros-copy and optical tweezers1. They

show that integral as well as GPI-anchored membrane proteins exhibita markedly reduced mobility in theaxon initial segment. Immobilizationrequires F-actin and might be medi-ated by tethering neuronal trans-membrane proteins to componentsof the spectrin–actin–ankyrin skeletonin the initial segment of the axon.

Thus, a new type of diffusion barrierdistinct from epithelial tight junctionssegregates the axon from the somato-dendritic membrane in neurons. Thisstudy paves the way for a detailedanalysis of the neuronal diffusion bar-rier, while it should also prompt othercell biologists to identify new types of

membrane barriers in other cell types.Several questions remain regardingthe exact mechanism of retention:how exactly are GPI-anchored orperipheral membrane proteinsretained within their membranedomains? What is the role of lipids, ifany, in this process? With the tech-niques in hand, we should have someanswers to these questions soon.

No diffusion beyond this point!

1 Winckler, B. et al. (1999) A diffusionbarrier maintains distribution ofmembrane proteins in polarizedneurons, Nature 397, 698–701

The human adult mammary glandductal system contains a layer ofepithelial cells surrounded by a layerof myoepithelial cells. These two celltypes were believed to have the sameectodermal stem-cell origin. However,the myoepithelial cell population canbe regenerated after depletion duringgestation and lactation periods. The

ratio between the two cell types shiftstowards the myoepithelial type inbenign tumours but towards theepithelial type in malignant tumours.These observations led to investigationof the cellular source of regenerationand/or conversion.

Using human mammary tissues,Péchoux et al.1 have shown that epi-

thelial cells can give rise to myoepi-thelial cells. They first purified the twocell types by using J5 antibodies againsta myoepithelial specific cell-surfacemarker (CALLA) to remove myoepithe-lial cells and MAM-6 antibodies for epi-thelial cells. The identities of these cellswere verified by the expression of cyto-keratin (CK18 and CK19) in epithelial

Keeping abreast of myoepithelial ontogeny

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trends in CELL BIOLOGY (Vol. 9) May 1999 173

cells, and vimentin, alpha-sm actin andb4 integrin in myoepithelial cells. Theyalso developed specific media, CDM4and CDM6, respectively, for culture of myoepithelial and epithelial cells.Furthermore, the two cell types remainstable even after a number of passagesas demonstrated by the constant ratio of expression of CK8 and CK14.After establishing the identities, theyswitched the culture media for the twocell types. In CDM6, the normal or theretroviral-immortalized myoepithelialcells remain the same. However, whenthe epithelial cells were put in CDM4,they changed morphologically, lost

expression of CK18 and CK19 andgained expression of vimentin, CALLAand alpha-sm actin. Their myoepithe-lial protein pattern was also confirmedby cellular sorting using b4 integrinexpression followed by two-dimen-sional gel electrophoresis analysis. Theauthors also looked for possible cellularconversion in vivo using doubleimmunofluorescence and confocalmicroscopy. With the vimentin andalpha-sm actin double staining, theyfound that some cells expressed onlyvimentin, and, with CK18 and vimentindouble staining, some cells expressedboth markers.

These results reveal the existence ofepithelial progenitors for myoepithelialcells. However, we still do not knowwhether the two cell types come froma common stem cell and what triggersor inhibits the conversion.

1 Péchoux, C., Gudjonsson, T.,Ronnov-Jessen, L., Bissell, M. J. andPetersen, O. W. (1999) Humanmammary luminal epithelial cellscontain progenitors to myoepithelialcells, Dev. Biol. 206, 88–99

One hundred years ago, J. Loebstated that ‘The ions, and not thenucleins, in the spermatozoon areessential to the process of fertilization(which may interest those whobelieve with me that physiologistsought to pay a little more attention toinorganic chemistry)’1. This sugges-tion is well heeded by those studyingegg activation, for we now know thatfertilization triggers a rise in intra-cellular Ca21, which is necessary andsufficient for the slow block topolyspermy and the onset of embry-onic development. Primarily, inositol(1,4,5)-trisphosphate (IP3) mediatesCa21 release, and a previous study2 instarfish eggs suggests that phospho-lipase C g (PLCg) mediates IP3 release.The question of whether PLCg isinvolved in egg activation in otherorganisms, and whether the fertiliz-ation-induced events, such as eggalkalinization, sperm entry, malepronuclear formation and entry intoS phase, are mediated by PLCg isaddressed by Carroll et al.3

Using a dominant–negative PLCg(tandem PLCg–SH2 domains), theauthors ask whether the fertilizationsignal-transduction pathway in seaurchin eggs requires PLCg activity.Injecting eggs with PLCg–SH2 blocksthe fertilization-induced Ca21 increase,egg alkalinization, the onset of S phaseand results in polyspermic fertilization.Co-injecting IP3 and PLCg–SH2 resultsin Ca21 release, suggesting that PLCgmight directly produce IP3, as instarfish. Interestingly, sperm entry andformation of the male pronucleus arenot blocked by injecting PLCg–SH2,indicating that a previously unrecog-nized Ca21-independent pathwayleads to these events. Of technicalinterest is the authors’ development ofa new technique to study DNA repli-cation, in which in vivo incorporationof a fluorescent nucleotide into newlyreplicated DNA can be observed usingconfocal microscopy.

Another recent study4 demonstratesthat fertilization of Xenopus eggs isblocked by inhibiting tyrosine kinase

activity and suggests that a Src-like pro-tein might be involved. It is tempting tospeculate that the factor upstream ofPLCg in echinoderms is a tyrosine kinasesince PLCg activation in other systemsrequires binding to, and phosphoryl-ation by, a tyrosine kinase. The findingthat Src might be involved in Xenopusegg activation will surely stimulaterelated studies in sea urchin eggs.

A century since Loeb

1 Loeb, J. (1899) Am. J. Phys. 3, 135–1382 Caroll, D. J., Ramarao, C. S., Mehlmann,

L. M., Roche, S., Terasaki, M. and Jaffe,L. (1997) J. Cell Biol. 138, 1303–1311

3 Carroll, D. J., Albay, D. T., Terasaki, M.,Jaffe, L. A. and Foltz, K. R. (1999)Identification of PLCg-dependent and independent events duringfertilization of sea urchin eggs, Dev. Biol. 206, 232–247

4 Glahn, D., Mark, S. D., Behr, R. K. andNuccitelli, R. (1999) Tyrosine kinaseinhibitors block sperm-induced eggactivation in Xenopus laevis, Dev. Biol.205, 171–180

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