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Cdk5, a member of the cyclin-dependent kinase family, has been shown to play an important role in development of the central nervous system in mammals when partnered by its activator p35. Here we describe the cloning and characterization of a novel activator of cdk5 inXenopus,Xp35.2. Xp35.2 is expressed during development initially in the earliest differentiating primary neurons in the neural plate and then later in differentiating neural tissue of the brain. This is in contrast to the previously describedXenopuscdk5 activator Xp35.1 which is expressed over the entire expanse of the neural plate in both proliferating and differentiating cells. Expression of both Xp35.1 and Xp35.2 and activation of cdk5 kinase occur when terminal neural differentiation is induced by neurogenin and neuro D overexpression but not when only early stages of neural differentiation are induced by noggin. Moreover, blocking cdk5 kinase activity specifically results in disruption and reduction of the embryonic eye where cdk5 and its Xp35 activators are expressed. Thus, cdk5/p35 complexes function in aspects of neural differentiation and patterning in the early embryo and particularly in formation of the eye.

Most of the mRNA sequences coding for alpha and beta tubulin in embryonic chick brain have been determined by sequencing of cloned cDNA copies of these mRNA copies of these mRNAs. From a 1,682-base pair cDNA sequence we have deduced the entire protein sequence for beta tubulin. For alpha tubulin, all but about 38 N-terminal amino acids have been deduced from the cDNA sequence. Although tyrosine has previously been shown to be post-translationally added to the C-terminus of alpha tubulin by a specific ligase, we conclude that the primary post-translational even must be the removal, not the addition of tyrosine because a terminal tyrosine is encoded by the mRNA.

Publisher Summary This chapter studies the role of small molecules or proteins in perturbing the association–dissociation steady state of actin and tubulin. It discusses the effect of molecules that bind to monomers at the ends of filaments, the so-called capping proteins. The chapter also discusses differences between large structures that interact with several monomers on filament ends and therefore remain attached during assembly and disassembly of monomers and proteins or factors that bind to single monomers on the ends of filaments and do not remain attached to the monomers. This analysis provides an indication of the range of what capping proteins and binding proteins can achieve. The models proposed in the chapter are illustrative rather than exhaustive, and in some cases they have been chosen to reflect as closely as possible the properties of known factors.

The Xenopus egg and embryo, throughout the transcriptionally inactive early cleavage period, were found to contain a store of approximately 8 X 10(8) molecules of the small nuclear RNA (snRNA) U1, sufficient for 4,000-8,000 nuclei. In addition, when transcription is activated at the twelfth cleavage (4,000 cell-stage), the snRNAs U1, U2, U4, U5, and U6 are major RNA polymerase II products. From the twelfth cleavage to gastrulation, U1 RNA increases sevenfold in 4 h, paralleling a similar increase in nuclear number. This level of snRNA transcription is much greater than that typical of somatic cells, implying a higher rate of U1 transcription or a greater number of U1 genes active in the embryo. The Xenopus egg also contains snRNP proteins, since it has the capacity to package exogenously added snRNA into immunoprecipitable snRNP particles, which resemble endogenous particles in both sedimentation coefficient and T1 RNase digestibility. SnRNP proteins may recognize conserved secondary structure of U1 snRNA since efficient packaging of both mouse and Drosophila U1 RNAs, differing 30% in sequence, occurs. The Xenopus egg and embryo can be used to pose a number of interesting questions about the transcription, assembly, and function of snRNA.

Nerve growth factor induces neurite process formation in pheochromacytoma (PC12) cells and causes the parallel increase in levels of the microtubule-associated proteins, tau and MAP1, as well as increases in tubulin levels. Mechanisms to insure balanced accumulation of microtubule proteins and make their levels highly responsive to nerve growth factor were investigated. The effects on tau, MAP1, and tubulin are due to changes in protein synthesis rates, which for tau and tubulin we could show are due in part to changes in the mRNA levels. Whereas tubulin shows feedback regulation to modulate synthesis up or down, tau protein synthesis is not affected in a straightforward way by microtubule polymerization and depolymerization. The degradation of tau, MAP1, and both tubulin polypeptides, however, are stimulated by microtubule depolymerization caused by colchicine, or nerve growth factor removal. Combined feedback on synthesis and stability make tubulin levels highly responsive to assembly states. In addition, the linkage of tau and MAP1 turnover with the state of microtubule polymerization amplifies any change in their rate of synthesis, since tau and MAP1 promote microtubule polymerization. This linkage lends itself to rapid changes in the state of the system in response to nerve growth factor.

Tau protein is a family of microtubule binding proteins, heterogeneous in molecular weight, that are induced during neurite outgrowth and are found prominently in neurofibrillary tangles in Alzheimer's disease. The predicted amino acid sequences of two forms of tau protein from mouse brain were determined from complementary DNA clones. These forms are identical in their amino-terminal sequences but differ in their carboxyl-terminal domains. Both proteins contain repeated sequences that may be tubulin binding sites. The sequence suggests that tau is an elongated molecule with no extensive alpha-helical or beta-sheet domains. These complementary DNAs should enable the study of various functional domains of tau and the study of tau expression in normal and pathological states.

We have produced extracts of frog eggs that can perform multiple cell cycles in vitro. Destruction of the endogenous messenger RNA arrests the extracts in interphase. The addition of exogenous cyclin mRNA is sufficient to produce multiple cell cycles. The newly synthesized cyclin protein accumulates during each interphase and is degraded at the end of each mitosis.

We have investigated the differences in microtubule assembly in cytoplasm from Xenopus oocytes and eggs in vitro. Extracts of activated eggs could be prepared that assembled extensive microtubule networks in vitro using Tetrahymena axonemes or mammalian centrosomes as nucleation centers. Assembly occurred predominantly from the plus-end of the microtubule with a rate constant of 2 microns.min-1.microM-1 (57 s-1.microM-1). At the in vivo tubulin concentration, this corresponds to the extraordinarily high rate of 40-50 microns.min-1. Microtubule disassembly rates in these extracts were -4.5 microns.min-1 (128 s-1) at the plus-end and -6.9 microns.min-1 (196 s-1) at the minus-end. The critical concentration for plus-end microtubule assembly was 0.4 microM. These extracts also promoted the plus-end assembly of microtubules from bovine brain tubulin, suggesting the presence of an assembly promoting factor in the egg. In contrast to activated eggs, assembly was never observed in extracts prepared from oocytes, even at tubulin concentrations as high as 20 microM. Addition of oocyte extract to egg extracts or to purified brain tubulin inhibited microtubule assembly. These results suggest that there is a plus-end-specific inhibitor of microtubule assembly in the oocyte and a plus-end-specific promoter of assembly in the eggs. These factors may serve to regulate microtubule assembly during early development in Xenopus.

Publisher Summary This chapter discusses the bioenergetics and kinetics of microtubule and actin filament assembly–disassembly. The chapter focuses on an important property of actin filaments and microtubules: their utilization of the free energy of adenosine triphosphate (ATP) or guanosine triphosphate (GTP) hydrolysis. To interpret the function of nucleoside triphosphate hydrolysis, a number of other properties are considered that include the structural and kinetic polarity of the filaments; their interaction at the ends with specific components; and the action of forces on the filaments. The important boundary conditions, such as forces acting in various ways on the filaments and the effect of materials that interact at the ends of filaments, are discussed in the chapter. The chapter also focuses on the biological implications of these properties in terms of capacity to do work, regulate length, and regulate spatial distribution.

We review the recent advances in understanding transitions within the cell cycle. These have come from both genetic and biochemical approaches. We discuss the phylogenetic conservation of the mechanisms that induce mitosis and their implications for other transitions in the cell cycle.

We have found that injection of bacteriophage lambda DNA into unfertilized Xenopus eggs causes the assembly around the DNA of structures resembling typical eucaryotic cell nuclei. These spherical structures begin to form 60-90 min after injection. They contain lambda DNA and are bounded by a phase-dense envelope. Immunofluorescent staining of the lambda-DNA-containing structures with anti-lamin antibody reveals the presence of the lamin nuclear proteins at the periphery of the structure, a pattern identical to that of embryonic nuclei. Electron microscopy reveals that the injected DNA is surrounded by a double bilayer nuclear membrane containing nuclear pore complexes. The "nuclei" containing lambda DNA respond to modulators of the Xenopus cell cycle in a manner that mimics the response of embryonic nuclei to these modulators during mitosis. These results suggest that nuclear reassembly and breakdown occur independently of specific DNA sequence information.

The mechanism of microtubule polymerization and depolymerization has been studied with protein purified from extracts of porcine brain. Under polymerizing conditions characteristic microtubules composed of parallel protofilaments are observed in the electron microscope. Under depolymerizing conditions three forms are observed: double rings of outside diameter 49 nm, spirals, and 7-nm globular subunits. Under the same conditions two boundaries are observed in the analytical ultracentrifuge at 6S and 36S, whether depolymerization is accomplished by cooling to 0°C, by addition of 1 mM CaCl2 at 25°C, or by removal of GTP. On polymerization all of the 36S and most of the 6S is converted to a fast-sedimenting form which the electron microscope reveals to be microtubules. The depolymerization mixture may be fractionated by gel chromatography into two fractions, one consisting solely of 6S and the other mostly 36S. Neither fraction regenerates the original equilibrium mixture. The 36S form may be reversibly dissociated into 6S subunits by addition of NaCl. From these and other considerations we have postulated that microtubule protein is composed of two different types of tubulin, both of which participate in polymerization. Studies are reported showing that colchicine does not dissociate microtubule rings but blocks polymerization by interfering with their proper lateral association into a protofilament array within microtubules. The role of GTP in polymerization is also discussed. Electron micrographic evidence is presented suggesting the conversion of protofilaments directly into rings and spirals, and a pathway for microtubule assembly is proposed.

Pattern formation and temporal control of gene expression in Xenopus development were investigated using fibronectin as a biochemical marker. We determined the spatial localization of fibronectin in the embryo by immunofluorescence and the temporal program of its expression by biosynthesis studies and Western blotting techniques. At the start of gastrulation, fibronectin is localized on the roof of the blastoccel which serves as the surface upon which mesodermal cells will migrate. However, since we find fibronectin secreted by all parts of the embryo, localization is probably achieved through spatially localized receptors that bind secreted fibronectin. Fibronectin levels and fibronectin synthesis rates increase following the midblastula stage. This increase is independent of transcription and therefore involves activation of maternal RNA for fibronectin. Since this message mobilization also occurs in activated but unfertilized eggs, this event must be regulated separately from the midblastula transition.

We report here that microtubules in vitro coexist in growing and shrinking populations which interconvert rather infrequently. This dynamic instability is a general property of microtubules and may be fundamental in explaining cellular microtubule organization.

The primary patterning event in early vertebrate development is the formation of the mesoderm and its subsequent induction of the neural tube. Classic experiments suggest that the vegetal region signals the animal hemisphere to diverge from the pathway of forming ectoderm to form mesoderm such as muscle. Here we show that bovine basic FGF has a limited capacity to induce muscle actin expression in animal hemisphere cells. This level of expression can be raised to levels normally induced in the embryo by another mammalian growth factor, TGF-beta, which by itself will not induce actin expression. We show that the Xenopus embryo contains an mRNA encoding a protein highly homologous to basic FGF. These results together with the identification of a maternal mRNA with strong homology to TGF-beta, suggest that molecules closely related to FGF and TGF-beta are the natural inducers of mesoderm in vertebrate development.

A heat stable protein essentail for microtubule assembly has been isolated. This protein, which we designate tau (tau), is present in association with tubulin purified from porcine brain by repeated cycles of polymerization. Tau is separated from tubulin by ion exchange chromatography on phosphocellulose. In the absence of tau, tubulin exists entirely as a 6S dimer of two polypeptide chains (alpha and beta tubulin) with a molecular weight of 120,000, which will not assemble into microtubules in vitro. Addition of tau completely restores tubule-forming capacity. Under nonpolymerizing conditions, tau converts 6S dimers to 36S rings-structures which have been implicated as intermediates in tubule formation. Hence, tau appears to act on the 6S tubulin dimer, activating it for polymerization. The unique ability of tau to restore the normal features of in vitro microtubule assembly makes it likely that tau is a major regulator of microtubule formation in cells.

Enucleated and nondividing amphibian eggs undergo cyclic changes in cell morphology and in the level of maturation promoting factor (MPF) with a period similar to the early cleavage cycle. We show here that there is a corresponding phosphorylation and dephosphorylation of specific proteins associated with this fundamental cell cycle. M-phase is associated with a general increase in phosphatase activity and specific phosphorylation of a small set of M-phase proteins, reflected in an increased stochiometry of phosphate and increased turnover. At the end of metaphase and correlated with a drop in MPF the phosphoproteins are rapidly lost. By microinjecting M-phase phosphoproteins into arrested interphase and metaphase eggs we could show that the specific M-phase phosphorylation was not due to specificity in phosphatase action. The ability to segregate synthesis from phosphorylation demonstrates that regulation is not on the level of synthesis of the M-phase proteins. Taken together these data suggest that regulation of kinase activity in M-phase in the face of general rapid phosphate turnover in the egg plays an important role in the regulation of the fundamental mitotic cycle.

The microtubule cytoskeleton is thought to be intimately involved in generating and maintaining cell polarity1,2 and can generate many different morphological structures from a few structural elements3. The mechanism by which these structures are generated has been partially elucidated from studies of microtubule polymerization both in vitro4–6 and in vivo7,8. Microtubules in vitro exist in growing (polymerizing) and shrinking (depolymerizing) populations that interconvert infrequently. This behaviour, termed dynamic instability4, permits microtubules in the cell rapidly to explore different arrangements and allows selective stabilization of specific morphologies9,10. To investigate the regulation of these processes, we have implemented techniques for direct observation of fluorescently labelled microtubules11 and developed them to observe the dynamic behaviour of individual microtubules in single living cells. Sammak and Borisy12 recently used this technique to show that the dynamics of microtubules in fibroblasts is explained by dynamic instability. Although we also conclude here that dynamic instability explains much of microtubule behaviour in vivo, we find significant deviations from the properties of tubulin in vitro. These results suggest that local cytoplasmic factors strongly influence microtubule dynamics; such control has important implications for cellular morphogenesis.

Depolymerization products of purified microtubules from porcine brain were examined by sedimentation analysis and electron microscopy. The complete depolymerization mixture exhibited 36S and 6S components in concentration-dependent equilibrium, whether depolymerization was caused by low temperature or by calcium ion. These components were recognized by electron microscopy as spirals and rings, and small particles. Agarose column chromatography yielded two major fractions, a leading one comprising mostly 36S and some 6S material and a trailing one of solely 6S material. The latter had high specific colchicine-binding activity and no tendency to polymerize. For the leading peak material these properties were the converse. It is proposed that tubulin molecules (of mass 110,000 daltons) exist in two states, here called X and Y, with those of the X-state equivalent to the material found predominantly in the trailing fraction, and those of the Y-state equated with the material in the leading fraction. Participation of tubulin molecules in microtubule assembly and disassembly is discussed, based on the assumption that those of both states have longitudinal and lateral binding domains whose strengths differentially depend upon temperature and calcium-ion concentration.

The midblastula transition (MBT) in Xenopus can be initiated prematurely by blocking the fundamental cell-cycle oscillator with cycloheximide, in which case motility and transcription are quickly initiated. Using various inhibitors of specific events of the cell cycle that do not inhibit the autonomous oscillator, we have shown that transcription is activated when DNA synthesis is interrupted and motility is activated when cell cleavage is inhibited. Furthermore, very low levels of transcription are found to occur before the MBT. These results demonstrate that the pre-MBT egg is fully competent for transcription and motility and suggest that different features of the rapid early cell cycle normally suppress these events.

We have isolated chromosomes from Chinese hamster ovary cells arrested in mitosis with vinblastine and examined the interactions of their kinetochores with purified tubulin in vitro. The kinetochores nucleate microtubule (MT) growth with complex kinetics. After an initial lag phase, MTs are continuously nucleated with both plus and minus ends distally localized. This mixed polarity seems inconsistent with the formation of an ordered, homopolar kinetochore fiber in vivo. As isolated from vinblastine-arrested cells, kinetochores contain no bound tubulin. The kinetochores of chromosomes isolated from colcemid-arrested cells or of chromosomes incubated with tubulin in vitro are brightly stained after anti-tubulin immunofluorescence. This bound tubulin is probably not in the form of MTs. It is localized to the corona region by immunoelectron microscopy, where it may play a role in MT nucleation in vitro.

By examining microtubule regrowth using immunofluorescence with antibody to tubulin, we have studied the structure and intracellular localization of microtubule initiation sites in undifferentiated and differentiated mouse neuroblastoma cells. The undifferentiated cells are round and lack cell processes. They contain an average of 12 initiation sites per cell. Each of these sites, which are located near the cell nucleus, initiates the growth of several microtubules in a radial formation. In contrast to the undifferentiated cells, neuroblastoma cells stimulated to differentiate by serum deprivation are asymmetrical, containing one or two very long neurites. These cells have a single, large microtubule initiation center which can be visualized not only by immunofluorescence but by phase-contrast and differential interference microscopy as well. The initiation site measures 3-4 mu in diameter and is located in the cell body along a line defined by the neurite. During cell differentiation, the large initiation, the large initiation center seems to be formed by the aggregation of many smaller sites. This process procedes neurite extension by about 24 hr. The growth of microtubules from this center appears to be highly oriented, since most microtubules initially grow into the neurite processes rather than into the cell interior. Thus major changes in the structure and location of microtubule initiation sites occur during the differentiation of neuroblastoma cells. Similar changes are likely to be involved in alterations in the morphology of other cell types.

Although numerous studies have suggested ways in which the assembly of cytoskeletal proteins can be regulated physiologically, less information has been generated on the regulation of the synthesis of these proteins. Ben-Ze'ev et al. recently suggested that the synthesis of tubulin in mouse 3T6 cells is affected by the state of assembly of microtubules. We have investigated the level at which this apparent modulation of tubulin synthesis takes place, using cloned cDNA probes for alpha- and beta-tubulin mRNAs to measure the amounts of tubulin mRNAs combined with immunoprecipitation of tubulin to monitor the rate of protein synthesis. We have found that in many, but not all, cell types tubulin synthesis decreases very rapidly in response to microtubule inhibitors that increase the monomer pool. This decline in synthesis is associated with decline in the amounts of both alpha- and beta-tubulin mRNAs. Kinetic studies of tubulin protein synthesis and RNA levels suggest that the tubulin monomer may regulate the rate of tubulin mRNA transcription. It is likely that tubulin synthesis can be shut off quickly in the cell as the result of short half-lives of the tubulin mRNAs, which may be as short as 1--2 hr. These data suggest that the cell exploits the instability of the tubulin mRNAs as a means to regulate precise levels of the monomer-tubulin pool.

We have microinjected biotinylated tubulin into mitotic fibroblast cells to identify the sites in the spindle at which new subunits are incorporated into microtubules (MTs). Labeled subunits were visualized in the electron microscope using an antibody to biotin followed by a secondary antibody coupled to colloidal gold. Astral MTs incorporate labeled subunits very rapidly by elongation of existing MTs and by new nucleation from the centrosome. At a slower rate, kinetochore MTs incorporate subunits at the kinetochore progressively during metaphase, suggesting a slow poleward flux of subunits in the kinetochore fiber. When cells injected in metaphase were examined in anaphase, a significant fraction of kinetochore MTs was unlabeled, suggesting that depolymerization had occurred at the kinetochore concomitant with chromosome to pole movement. The existence of opposite fluxes at the kinetochore during metaphase and anaphase suggests that two separate forces are responsible for chromosome congression and anaphase movement.

Publisher Summary This chapter presents recent studies on in vitro assembly, nucleation, and growth of microtubules along with the properties that shed light on their biological role. The biochemical study of structural proteins such as tubulin and actin is a new area. It has received a considerable amount of attention recently which, it is hoped, reflects progress. Despite the fact that actin is in general the most common cellular protein and that so much was known about it from studies of muscle, not until recently has its role in nonmuscle cells been appreciated. Tubulin, perhaps the second most common intracellular protein, was not described until the 1960s. Functional studies of tubulin assembly were not performed until 1972. The means of regulation and the principles of self-assembly are also presented in the chapter. It discusses the experiments that (1) emanated from the discovery of conditions for assembly of microtubules from brain extracts, (2) relate to the biochemical properties of tubulin, and (3) that utilize in vitro systems for studying nucleation and growth of cytoplasmic microtubules. Certain topics in microtubule assembly have been well studied in the past few years

The nuclear lamina of vertebrates is composed of several major polypeptides that range in mol. wt from 60 to 80 kd. In mammals, the three major lamin proteins are designated A, B and C. Two major lamins have been described in Xenopus somatic tissues; two other lamins are expressed primarily in germ cells. We have analysed a cDNA clone encoding a Xenopus lamin that is highly homologous to human lamins A and C. The predicted protein has the carboxy-terminal domain characteristic of human lamin A and is thus a lamin A homologue. Surprisingly, the lamin encoded by the cDNA clone is not one of the known Xenopus lamins. The encoded protein is distinct in size from the oocyte lamin LIII and the two somatic lamins LI and LII. Monoclonal antibodies specific for LII, LIII and LIV (the lamin of male germ cells) do not recognize the protein encoded by the cDNA clone; conversely, a polyclonal antibody against the encoded protein does not recognize any of the known Xenopus lamins. This lamin is expressed late in embryonic development, and is present in all adult somatic cells examined, except erythrocytes. Thus frogs and mammals are similar in having three major somatic lamins that fall into distinct structural classes.

Publisher Summary This chapter provides information on the purification method of aspartate transcarbamoylase. The method described is slightly modified from Kirschner's method and provides high yields of purified catalytic and regulatory subunits. The procedure presented is analogous to that of Gerhart and Holoubek except that neohydrin is used in place of p-mercuribenzoate for the dissociation of aspartate transcarbamoylase (ATCase) and diethylaminoethyl (DEAE)-cellulose is used for the chromatographic separation of the subunits instead of DEAE-Sephadex. Aspartate transcarbamoylase catalyzes the first reaction unique to pyrimidine biosynthesis; in E. coli regulation is achieved in part by feedback inhibition of the enzyme by cytidine triphosphate (CTP), an end-product of the pathway. The purified enzyme when treated with the mercurial, pmercuribenzoate is known to dissociate into two types of subunits, one responsible for catalysis, termed “the catalytic subunit” and the other that binds both the inhibitor, CTP and the activator, adenosine triphosphate (ATP).

We constructed complexes between isolated chromosomes and microtubules made from purified tubulin to study the movement of chromosomes towards the 'minus' end of microtubules in vitro, a process analogous to the movement of chromosomes towards the pole of the spindle at anaphase of mitosis. Our results show that the energy for this movement is derived solely from microtubule depolymerization, and indicate that anaphase movement of chromosomes is both powered and regulated by microtubule depolymerization at the kinetochore.

In many mammalian cell types, increases in the level of nonpolymerized tubulin cause an inhibition in tubulin synthesis which is accompanied by a decrease in tubulin mRNA levels. To see whether inhibition is caused by nuclear or cytoplasmic events, two groups have recently examined the ability of enucleated cells to autoregulate tubulin synthesis.1,2 These experiments have demonstrated that transcription, processing, and transport of tubulin mRNAs from the nucleus to the cytoplasm are not major sites of autoregulation. Instead, monomeric tubulin must reduce, either directly or indirectly, the translatability of its own message.

Publisher Summary This chapter discusses the initiation of the cell cycle and establishment of bilateral symmetry in Xenopus egg. In the establishment of the cell cycle, in unfertilized Xenopus laevis eggs the cell cycle is arrested at second meiotic metaphase where no DNA synthesis is taking place. The period after fertilization in amphibian eggs is concerned with the completion of meiosis, fusion of the pronuclei, restarting the cell cycle, and the establishment of the dorsal–ventral asymmetry of the future embryo. Directly after fertilization there are a number of cortical and internal movements that are related to these events. Fertilized Xenopus eggs when rotated, sperm entrance point (SEP) down acquire the same yolk configuration as do unrotated eggs, that is, vitelline wall on the dorsal side, whereas Xenopus eggs rotated with the SEP up, acquire a mirror image configuration of vegetal yolk. Thus, the rearrangement of yolk correlates well with the reversal of the axis orientation by gravity, indicating a role for the yolk.

The messenger RNAs coding for alpha- and beta-tubulin have been isolated from embryonic chick brain. Although the mRNAs for the two tubulin subunits have been resolved on native gels, they are very similar in molecular weight (650,000 daltons) as judged by mobility on denaturing gels containing methy mercury. The mRNAs for beta- and gamma-actin have also been resolved on native gels, but migrate as an unresolved peak (molecular weight 6500,000-700,000 daltons) under denaturing conditions. Since the nonmuscle actins are substantially smaller proteins than alpha- and beta-tubulin, the large size of chick nonmuscle actin mRNAs suggests an unusually long untranslated region. Since tubulin and actin polypeptides are internal structural proteins, one would expect them to be synthesized only on free polysomes. Translation of mRNA derived directly from a purified membrane fraction or by puromycin release from that fraction, however, showed the synthesis of a small proportion of these proteins on polysomes that are membrane-associated. Peptide mapping has in all cases confirmed the identity of the products of cell-free synthesis with authentic alpha-tubulin, beta-tubulin and actin. Approximately 67% of the alpha- and 13% of the beta-tubulin chains produced by in vitro translation are competent for co-assembly into microtubules with added carrier microtubule protein.

tau protein isolated from porcine brain microtubules was further purified by electrophoretic elution from polyacrylamide gels and used to prepare antisera in rabbits. The antiserum to tau specifically stains mitotic spindles and a filamentous network within mouse fibroblasts when the indirect immunofluorescence technique is used. The staining of the filamentous network and mitotic spindles is identical to that observed when cells are treated with antiserum prepared against electrophoretically purified tubulin. The filamentous network observed with either serum is sensitive to Colcemid. Absorption of anti-tau serum with electrophoretically purified tubulin does not remove the immunofluorescent staining of the mitotic spindle, whereas absorption with electrophoretically purified tau protein does. Conversely, absorption of antitubulin serum with tubulin eliminates its ability to stain the mitotic spindle, whereas absorption with tau has no effect. We conclude that tau protein and tubulin are antigenically distinct proteins and that tau is an integral part of microtubules in vivo. These results also provide evidence that tau protein, or an antigenically related protein, is associated with microtubules not only in brain but also in other cell types.

Maturation-promoting factor (MPF) is a partially purified M-phase-specific activity that induces meiosis in frog oocytes and is detectable in mitotic lysates from cells of wide phylogenetic origins. We show here that without protein synthesis, addition and removal of MPF can drive the mitotic cycle in frog eggs, including nuclear membrane breakdown and reformation, chromosome condensation and decondensation, and suppression and initiation of DNA replication on endogenous DNA and on injected plasmid templates. We have also studied M-phase arrest induced by injection of unfertilized egg cytoplasm and show that this arrest blocks an endogenous cytoplasmic cell-cycle oscillator and causes the stabilization of MPF activity. The oscillator can be restarted by injection of Ca++, which causes chromosome decondensation, reinitiation of DNA replication, and loss of MPF activity. We have looked in more detail at how DNA replication responds to the level of MPF and show that the effects are on the chromatin template and not the replication machinery. These results suggest that in Xenopus embryos cell-cycle events of the nucleus, including DNA replication and mitosis, are controlled by the level of MPF activity, which is driven by or may be part of an autonomous cell-cycle oscillator. The way in which a more complicated somatic cell cycle may arise from the simple embryonic cell cycle is discussed.

The kinetics of microtubule assembly from purified porcine brain tubulin were examined by quantitative electron microscopy. Assembly at 37°C was initiated by the addition of GTP to 1 m m to the protein solution at ∼1 mg/ml in ammonium acetate buffer, pH 6·4. A solution of partially polymerized protein was mixed with a suspension of tomato bushy stunt virus at known particle concentration, and spray-deposited, in negative stain, upon specimen films. The resulting drop patterns were micrographed and the percent protein seen in distinguishable polymeric forms was calculated from the numbers, lengths, and known masses and mass/unit lengths of these entities. Clumping of protein by the uranyl acetate stain was avoided by use of a dual nebulizer which mixed protein and stain only upon spray droplet formation. The micrographs displayed ring-like forms, microtubules, and aggregates of parallel protofilaments—arrayed as flat, curved, and helical ribbons. Protein found as rings rapidly decreased from an initial 30% to a negligible amount at 2 minutes; the amount as microtubules increased asymptotically to an equilibrium value of 50% by 8 minutes; the quantity of ribbons was largest at 3 minutes (∼20%), but vanishingly small by 8 minutes. The results show that the fully formed microtubules consisted of protein which was initially both in the ring form and in the tubulin-dimer (6 S) form. They also quantitatively demonstrate that the twisted ribbons are assembly intermediates, those with a full protofilament complement presumably converting to microtubules by cylindrical folding.

We have used cloned chicken cDNA sequences for alpha- and beta-tubulin to investigate tubulin gene organization in Drosophila melanogaster. Experiments using genomic Drosophila DNA from several sources indicate that there are at least four copies each of the alpha-tubulin gene and the beta-tubulin gene. In situ hybridization experiments show that both the alpha- and beta-tubulin multigene families have dispersed arrangements on the chromosome. Genes for alpha-tubulin have been localized at chromosomal bands 67C, 84B/C, 84D and 85E, while genes for beta-tubulin have been detected at bands 60A/B and 85D. alpha-Tubulin and beta-tubulin chicken cDNA sequences can be used to select a specific mRNA species from a complex mixture which translates in vitro into alpha- or beta-tubulin protein. RNA blot hybridization using the cloned chicken cDNA sequences as probes shows that the alpha- and beta-tubulin messages detected are clearly different in length, with the message for alpha-tubulin measuring approximately 2000 bases and the message for beta-tubulin containing approximately 1800 bases.

Since the time of the great library of Alexandria, scholars have recognized the value of central repositories of knowledge. As scientists, we are particularly dependent on ready and unimpeded access to our published literature, the only permanent record of our ideas, discoveries, and research results, upon which future scientific activity and progress are based. The growth of the Internet is changing the way we access this literature, as more scientific journals produce online editions to supplement or replace printed versions. We urge journal publishers, their editors, and all working scientists to join together to create public, electronic archives of the scientific literature, containing complete copies of all published scientific papers.

• We studied serum samples from 106 patients, including 80 in the scleroderma spectrum, by indirect immunofluorescent microscopy, using PtK, rat kangaroo tissue culture cells as substrate. Anticentromere (Kinetochore) antibodies were present in 28 patients, and anticentriole antibodies were present in four patients. Anticentromere antibodies were usually present in patients with a benign, chronic form of systemic scleroderma, which has been termed the CREST (Calcinosis, Raynaud's phenomenon, esophageal involvement, sclerodactyly, and telangiectasia) syndrome. The four patients with a previously undescribed anticentriole antibody were all in the scleroderma spectrum. Possibly, these antibodies may have diagnostic and prognostic importance. Further, they will be useful in studying the structure and function of these cellular organelles. ( Arch Dermatol 1983;119:560-566)

Comparable processes in different species often involve homologous genes. One question is whether the network structure, in particular the feedback control structure, is also conserved. The bacterial chemotaxis pathways in E. coli and B. subtilis both regulate the same task, namely, excitation and adaptation to environmental signals. Both pathways employ many orthologous genes. Yet how these orthologs contribute to network function in each organism is different. To investigate this problem, we propose what is to our knowledge the first computational model for B. subtilis chemotaxis and compare it to previously published models for chemotaxis in E. coli. The models reveal that the core control strategy for signal processing is the same in both organisms, though in B. subtilis there are two additional feedback loops that provide an additional layer of regulation and robustness. Furthermore, the network structures are different despite the similarity of the proteins in each organism. These results demonstrate the limitations of pathway inferences based solely on homology and suggest that the control strategy is an evolutionarily conserved property., Computational modeling reveals some important differences in the networks that regulate chemotaxis in E. coli and B. subtilis, differences that are hard to predict on the basis of sequence homology alone