Membrane Specialization

Regulating Polarity in Pollen Tube Growth

Elongating at a rate of several micrometers per minute, pollen tubes are among the fastest growing cells known and are a good system for studying the development of cellular polarity. The pollen tube is believed to extend by a process of tip growth, in which secretory vesicles fuse with the growing tip of the tube to deliver cell membrane and cell wall material. The mechanism by which polarity is established in these cells has remained obscure, though a calcium gradient at the growing tip appears to have an important role (Pierson E.S., D.D. Miller, D.A. Callaham, J. van Aken, G. Hackett, P.K. Hepler. 1996. Dev. Biol. 174:160– 173). Work reported by Kost et al. (page ) provides the first direct evidence that a Rac-type small GTPase controls polar pollen tube growth by stimulating compartmentalized synthesis of phosphatidylinositol 4, 5-bisphosphate (PIP2).

After cloning At-Rac2, a Rac-like GTPase from Arabidopsis that is preferentially expressed in pollen and growing pollen tubes, Kost et al. constructed mutant forms of the gene that they predicted would be dominant negative or constitutively active. Expressing the dominant negative At-Rac2 in an in vitro model of pollen tube growth prevents tube elongation, whereas expression of the constitutively active form causes depolarized growth, resulting in formation of spherical balloons instead of elongating tubes. The protein copurifies with a phosphatidylinositol kinase (PIP-K) that produces PIP2, and GFP fusion constructs demonstrate that At-Rac2 localizes to the pollen tube tip.

The results support a model in which Rac stimulates PIP-K to produce a local concentration of PIP2 at the tip of the pollen tube. Although it has not been directly demonstrated yet, this pool of PIP2 might be hydrolyzed by phospholipase C to inositol 1, 4, 5-trisphosphate to produce the previously observed calcium gradient, or PIP2 may act directly as an effector. Benedikt Kost, first author on the study, suggests that “a similar mechanism could account for spatially and also temporally restricted membrane lipid-mediated signaling in other cell types.”

Rho-Kinase and Cell Motility

Rho, a small GTPase related to Rac, appears to have a key role in another type of membrane specialization, as Fukata et al. explain starting on page 347. Although it was known that Rho could regulate membrane ruffling and cell motility, the signal cascade for this process was not well understood. The same laboratory had previously found that adducin, which binds to F-actin filaments to promote the formation of a spectrin–actin mesh beneath plasma membranes, is a substrate of Rho-kinase in vitro, a downstream effector of Rho (Kimura, K., Y. Fukata, Y. Matsuoka, V. Bennett, Y. Matsuura, K. Okawa, A. Iwamatsu, and K. Kaibuchi. 1998. J. Biol. Chem. 273:5542–5548). Now Fukuta et al. elucidate the details of Rho-kinase activity on adducin in vivo and show that phosphorylation of adducin through the Rho/Rho-kinase pathway is crucial for membrane ruffling and cell motility.

Once they had determined the sites at which Rho-kinase phosphorylates adducin, Fukuta et al. prepared an antibody that specifically recognizes phosphorylated adducin and found that the phosphorylated form accumulates in the membrane ruffling area of Madin–Darby canine kidney (MDCK) epithelial cells treated with TPA. Dominant-negative mutant adducin and inhibitors of Rho and Rho-kinase all inhibit the TPA-induced membrane ruffling, and this effect is reversed by a constitutively active mutant of adducin. In a model system for wound healing, NRK49F cells, inhibition of Rho and dominant-negative mutant adducin inhibit cell migration. The constitutively active mutant adducin also inhibits cell migration, though it does not prevent membrane ruffling, suggesting that motility requires cycling between phosphorylated and dephosphorylated states.

The results point to a model in which Rho-kinase, activated by Rho, phosphorylates adducin, which then binds to F-actin to remodel the cytoskeleton and produce membrane ruffling and cell migration. The mechanism by which adducin produces these changes remains unknown.

A Dystrophin-related Complex in Epithelial Cells

The dystrophin-associated protein complex (DAPC) is a feature of membrane specialization sites in the neuromuscular junction and central nervous system synapses, and may have an important structural role in protecting muscle cells from shearing forces during contraction. Reasoning that the complex may also be important in epithelial tissue, Kachinsky et al. (page ) studied the localization of a dystrophin-related complex in MDCK cells.

Utrophin, a homologue of dystrophin, is present at the basolateral membrane of MDCK cells, and GFP tagging experiments show that syntrophin, an adapter protein that recruits other gene products to the DAPC, also localizes to the basolateral membrane. Syntrophin consists primarily of protein–protein interaction domains, including two pleckstrin homology (PH) domains, a PDZ domain, and a syntrophin unique (SU) domain. Using a series of truncated syntrophin-GFP constructs, Kachinsky et al. demonstrate that the second PH domain and the adjacent SU domain are sufficient to direct syntrophin to the basolateral membrane. Coimmunoprecipitation confirms that syntrophin is bound in a complex with utrophin, and the stoichiometry of the complex appears to be 2 syntrophin/1 utrophin/1 dystrobrevin, the same stoichiometry believed to be present in the muscle cell DAPC. But because the proteins subsequently recruited to the DAPC may vary from one cell type to another, Amy Kachinsky, first author on the paper, says that “it is interesting to postulate that this complex will have additional roles in epithelia that have not been characterized in muscle.”

Although utrophin appears to recruit syntrophin to the basolateral membrane, it is unclear what initially controls the targeting of utrophin in this system. Kachinsky et al. are currently examining the possibility that dystroglycan, which is found in the basolateral membrane of MDCK cells and is known to interact with dystrophin family proteins, might be responsible.

Impact of Telomere Cap Deregulation

On page , Smith and Blackburn present strong support for a view of telomere regulation in which the structure of the telomere cap, determined by the most terminal nucleic acid repeats, is at least as important as telomere length. Elizabeth Blackburn, senior author on the study, explains that “this is quite a change from what people had thought before, when length was imagined to be the important parameter of telomeres, all other things being equal.”

The researchers studied the budding yeast Kluveromyces lactis, in which the telomeres contain a precise 25-bp repeat sequence, allowing the creation of mutations with restriction site changes that can be traced through multiple generations. Mutations in ter1 that alter the repeat sequence in a way that affects binding of the Rap1p protein result in dramatic elongation of telomeres and also the loss of homogeneity in telomere length, producing smearing on a gel of telomeric DNA. Previous work (Krauskopf, A., and E.H. Blackburn. 1996. Nature. 383:354–357) showed that the most terminal repeat sequences were crucial for regulating telomere length, but the current report demonstrates the effects of telomere deregulation on cell morphology and DNA content, independent of telomere length.

Under the microscope, populations of ter1 mutants contain a high proportion of morphologically abnormal cells compared with wild-type cultures. These monster cells appear grossly enlarged or elongated, suggesting a budding or division defect. FACS® analysis also shows an increase in the number of cells with greater than diploid DNA content. To verify the importance of capping, the researchers recapped the telomeres of ter1 mutants with functionally wild-type repeats after many generations with deregulated caps. This reestablished regulation, causing telomeric DNA to migrate in discrete bands rather than a smear, but the telomeres remained elongated. Despite their longer telomeres, these recapped strains show wild-type morphology and DNA content.

It is unclear exactly why telomere cap deregulation leads to the production of monster cells, but the phenotype suggests that DNA missegregation is important. And while K. lactis provides a convenient system for studying the phenomenon, Blackburn stresses that the findings are likely to have broader implications: “What we and others have discovered about telomeres in yeasts have turned out to be fundamental properties of telomeres in general, and there is no reason to think this will be an exception.”

Physical Structure of Mitotic Chromosomes

Chromosome condensation is critical for proper cell division, and the physical structure of condensed mitotic chromosomes has been the subject of extensive speculation. Unfortunately, no definitive experimental technique has been available to test models of chromosome structure, which have included hierarchical folding of chromatin, loops attached to a central scaffold, and a cross-linked unorganized gel. Houchmandzadeh and Dimitrov (page ) now report the measurement of two types of elasticity in condensed chromosomes assembled in vitro, demonstrating that the chromosomes consist of a thin, rigid core surrounded by a soft envelope.

Using specially designed micropipettes, the authors determined the force required to stretch a chromosome lengthwise, then used this result to calculate the bending flexibility the chromosome would have if it were a gel-like material or a hierarchically folded helix. Chromosome flexibility was then empirically quantified by observing the fluctuation of chromosome shape under the microscope at a set temperature. The experiment shows that the chromosomes are ∼2,000-fold more flexible than predicted for a gel-like or hierarchically folded structure, suggesting that a rigid core acts to inhibit stretching while permitting bending. Similar elastic behavior has been observed in titin, a protein that has been found associated with mitotic chromosomes, suggesting that a titin-like non-histone backbone acts as a scaffold for these chromosomes.

Although the current report focuses on chromosomes assembled in vitro, a series of biophysical measurements shows that these chromosomes closely mimic the in vivo situation. Applying the same measurement techniques to chromosomes inside living cells raises considerable technical challenges, but Bahram Houchmandzadeh, first author on the study, explains that “the hope is that as we learn more and more on simple systems, we can investigate more complex systems.”

By Alan W. Dove, 712 W. 176th St. #2A New York, NY 10033-7502. E-mail: a.dove@erols.com