Fifteen formins for an actin filament: a molecular view on the regulation of human formins.

The regulation of the actin cytoskeleton is a key process for the stability and motility of eukaryotic cells. Besides the Arp2/3 complex and its nucleation promoting factors, WH2 domain-containing proteins and a diverse family of formin proteins have recently been recognized as actin nucleators and potent polymerization factors of actin filaments. Formins are defined by the presence of a catalytic formin homology 2 (FH2) domain, yet, the modular domain architecture appears significantly different for the eight formin families identified in humans. A diverse picture of protein localization, interaction partners and cell specific regulation emerged, suggesting various functions of formins in the building and maintenance of actin filaments. This review focuses on the domain architecture of human formins, the regulation mechanisms of their activation and the diversity in formin cellular functions.

Mechanism and function of formins in the control of actin assembly.

Formins are a widely expressed family of proteins that govern cell shape, adhesion, cytokinesis, and morphogenesis by remodeling the actin and microtubule cytoskeletons. These large multidomain proteins associate with a variety of other cellular factors and directly nucleate actin polymerization through a novel mechanism. The signature formin homology 2 (FH2) domain initiates filament assembly and remains persistently associated with the fast-growing barbed end, enabling rapid insertion of actin subunits while protecting the end from capping proteins. On the basis of structural and mechanistic work, an integrated model is presented for FH2 processive motion. The adjacent FH1 domain recruits profilin-actin complexes and accelerates filament elongation. The most predominantly expressed formins in animals and fungi are autoinhibited through intramolecular interactions and appear to be activated by Rho GTPases and additional factors. Other classes of formins lack the autoinhibitory and/or Rho-binding domains and thus are likely to be controlled by alternative mechanisms.

High expression of the gamma5 isoform of G protein in neuroepithelial cells and its replacement of the gamma2 isoform during neuronal differentiation in the rat brain.

High concentrations of G proteins, which include multiple isoforms of each subunit, alpha, beta, and gamma, are expressed in the adult brain. In this study, we concentrated attention on changes of these isoforms during embryonic development in the rat brain. Concentrations of gamma2 as well as GoAalpha, GoBalpha, and beta2 were low in early embryogenesis and then increased, whereas expression of gamma5, in contrast, was initially high followed by a drop, with only very low levels observed throughout postnatal development. Among the other isoforms, Gi1alpha, G(s)alpha-short, G12alpha, G13alpha, beta4, gamma3, gamma7, and gamma12 were present in the embryonic brain at low levels, but their levels markedly increased after birth. In contrast, the levels of Gi2alpha, G(s)alpha-long, Gq/11alpha, and beta1 were essentially constant throughout. Immunohistochemical staining of the brain vesicles in the embryos showed gamma5 to be specifically expressed in the proliferative region of the ventricular zone, whereas gamma2 was mainly present in differentiated neuronal cells of the marginal zone. Furthermore, differentiation of P19 mouse embryonal carcinoma cells to neuronal cells with retinoic acid induced the expression of gamma2 and a decrease of gamma5, the major isoform in the undifferentiated state. These results suggest that neuronal differentiation is responsible for the on/off switch of the expression of gamma2 and gamma5 subunits.

Epsilon subunit-containing acetylcholine receptors in myotubes belong to the slowly degrading population.

Two types of muscle acetylcholine receptors (AChRs) can be distinguished on the basis of their degradation rates and sensitivities to innervation, muscle activity, and agents elevating intracellular cAMP. The first type (Rs), is present in a stable form (degradation t1/2 = approximately 10 d) at the adult innervated neuromuscular junctions (NMJs). Rs can also exist in a less stable form (called accelerated Rs; t1/2 = approximately 3-5 d) at denervated NMJs and in aneurally cultured myotubes; agents that increase intracellular cAMP reversibly modulate Rs stability. The second type of AChR is a rapidly degrading receptor (Rr) expressed only in embryonic and noninnervated muscles. Rr can be stabilized by ATP and not by cAMP. This study tested the hypothesis that the degradation properties unique to the Rs are attributable to the presence of the epsilon subunit. Immunoprecipitation and Western blot analysis of AChRs extracted from rat muscle cells in tissue culture showed that AChRs recognized by antibodies against the epsilon subunit degraded as a single population with a half-life similar to that of the slow component, Rs, in these cells. In addition, as for Rs receptors in denervated NMJs and cultured muscle cell, the degradation rate of these epsilon-containing AChRs was stabilized by dibutyryl-cAMP. The data indicate that the epsilon-containing AChRs behave like Rs. Thus, the presence of the epsilon subunit is sufficient for selecting an AChR molecule to the Rs pool.

Fibronectin isoform distribution in the mouse. I. The alternatively spliced EIIIB, EIIIA, and V segments show widespread codistribution in the developing mouse embryo.

Fibronectins (FNs) are extracellular matrix glycoproteins that are essential for embryonic development. In order to gain clues to possible developmental roles played by the particular isoforms of FN, we used indirect immunofluorescence microscopy to examine and compare the distributions of the alternatively spliced EIIIB, EIIIA, and V segments, as well as the total pool of FNs, in serial sections from mouse embryos. Antibodies to each of these segments produced staining patterns that colocalized during gastrulation (E7.5) and during early morphogenesis of somites and notochord (E9.5). During the period of continuing organogenesis in the latter half of gestation (E10.5 to E16.5), the antibodies generally continued to produce similar staining patterns localized to epithelial basement membranes, stromal connective tissues, blood vessel walls, and muscles. However, as development proceeded, there was a gradual decline in the intensity of staining for the spliced segments relative to the total pool of FN, with a particularly noticeable decline in staining for EIIIB and EIIIA segments in certain glandular organs, including the liver. A specific reduction in expression of these latter two segments was also evident in the uterus and placenta at early timepoints in gestation. However, the most dramatic difference in the expression of the spliced segments occurred in developing hyaline cartilage, which showed a selective reduction in staining for the EIIIA segment that was evident in the axial skeletal precursors by E12.5 and complete throughout the embryo by E15.5. Our findings suggest that the alternatively spliced EIIIB, EIIIA, and V segments are included in the FN that is required for the morphogenesis of "FN dependent" structures, including somites, notochord, and the vasculature. Conversely, these segments would appear to play divergent, and sometimes exclusive, biological roles in specific tissues such as liver, cartilage, and placenta.

The typing of fetal antigen 2 in human amniotic fluid.

Fetal antigen 2 (FA-2) has been identified in amniotic fluid and shown to be of fetal origin. In this study we have extended previous observations on FA-2 heterogeneity with respect to both size and charge using gel filtration, ion-exchange chromatography, non-dissociating polyacrylamide gel electrophoresis and sodium dodecyl sulphate polyacrylamide gel electrophoresis. From the diversity of forms we have been able to define two principal FA-2 types, type A and type B. Type A has a high molecular mass (140 kDa), has subunits of 33 kDa and 29 kDa, and elutes at approximately 0.27 mol/l sodium chloride from diethylaminoethyl (DEAE)-Sephacel. Type B has the same mass and subunits as type A, but elutes at approximately 0.24 mol/l sodium chloride from DEAE-Sephacel. Other low molecular mass forms of FA-2 have also been identified. All FA-2 forms described were shown to be common to all amniotic fluid samples studied and were not attributable to artefacts of collection or storage. It was also demonstrated that the recently described FA-2 RIA is specific for FA-2 types A and B and the conversion of arbitrary units FA-2 into micrograms applies to type A. The typing is discussed with respect to (i) the aminopropeptide of the alpha 1 chain of human procollagen type I, (ii) the 24 kDa phosphoprotein in developing bone and (iii) fetal calf ligament protein 1 (FCL-1), suggesting that they are the same protein.