Recruitment of the γ-Tubulin Ring Complex to Drosophila Salt-stripped Centrosome Scaffolds

Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and γ-tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional centrosome components required for nucleation, we have developed an in vitro complementation assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila embryo extract. The CP60 and CP190 can be removed from this extract without effect, whereas removing the γ-tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, γ-tubulin is found in two distinct protein complexes of 240,000 and ∼3,000,000 D. The larger complex, which is analogous to the Xenopus γ-tubulin ring complex (γTuRC) (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578–583), is necessary but not sufficient for complementation. An additional factor found in the extract is required. These results provide the first evidence that the γTuRC is required for microtubule nucleation at the centrosome.     

Human 76p: A new member of the gamma-tubulin-associated protein family

The role of the centrosomes in microtubule nucleation remains largely unknown at the molecular level. gamma-Tubulin and the two associated proteins h103p (hGCP2) and h104p (hGCP3) are essential. These proteins are also present in soluble complexes containing additional polypeptides. Partial sequencing of a 76- kD polypeptide band from these complexes allowed the isolation of a cDNA encoding for a new protein (h76p = hGCP4) expressed ubiquitously in mammalian tissues. Orthologues of h76p have been characterized in Drosophila and in the higher plant Medicago. Several pieces of evidence indicate that h76p is involved in microtubule nucleation. (1) h76p is localized at the centrosome as demonstrated by immunofluorescence. (2) h76p and gamma-tubulin are associated in the gamma-tubulin complexes. (3) gamma-tubulin complexes containing h76p bind to microtubules. (4) h76p is recruited to the spindle poles and to Xenopus sperm basal bodies. (5) h76p is necessary for aster nucleation by sperm basal bodies and recombinant h76p partially replaces endogenous 76p in oocyte extracts. Surprisingly, h76p shares partial sequence identity with human centrosomal proteins h103p and h104p, suggesting a common protein core. Hence, human gamma-tubulin appears associated with at least three evolutionary related centrosomal proteins, raising new questions about their functions at the molecular level.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

Differing requirements for Augmin in male meiotic and mitotic spindle formation in Drosophila

Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and sperm-producing male meiotic spermatocytes use centrosome-dependent and acentrosomal spindle-forming mechanisms. Here, we characterize the largely undefined, centrosome-independent spindle formation pathway used during male meiosis. Our live and fixed cell analyses of Drosophila spermatocytes reveal that acentrosomal microtubules are nucleated at kinetochores and in the vicinity of chromatin and that together these assemble into functional spindles. Mutational studies indicate that γ-tubulin and its extra-centrosomal targeting complex, Augmin, are vital for this process. In addition, Augmin facilitates efficient spindle assembly in the presence of centrosomes. In contrast to the pronounced recruitment of Augmin on spindles in other cell types, the complex is absent from those of spermatocytes but does accumulate on kinetochores. Polo kinase facilitates this kinetochore recruitment while inhibiting Augmin''s spindle association, and this in turn dictates γ-tubulin distribution and spindle density. Polo''s negative regulation of Augmin in male meiosis contrasts with its requirement in loading Augmin along mitotic spindles in somatic Drosophila cells. Together our data identify a novel mechanism of acentrosomal spindle formation in spermatocytes and reveal its divergence from that used in mitotic cells.     

Three-Dimensional Reconstruction of the Mammalian Centriole from Cryoelectron Micrographs: The Use of Common Lines for Orientation and Alignment

The microtubule organizing center of the animal cell (S. D. Fulleret al.,1992,Curr. Opin. Struct. Biol.2,264–274; D. M. Gloveret al.,1993,Sci. Am.268,62–68; E. B. Wilson, 1925), (The Cell in Development and Heredity) comprises two centrioles and the pericentriolar material. We have completed several three-dimensional reconstructions of individual centrioles from tilt series of cryoelectron micrographs. The reconstruction procedure uses minimization of the common lines residual to define the orientation of the centriolar ninefold symmetry axis and then uses this symmetry to generate a structure by weighted backprojection to 28-nm resolution. Many of the features of these reconstructions agree with previous, conventional transmission electron microscopy studies (M. Paintrandet al.,1992,J. Struct. Biol.108,107–128). The microtubule barrel of the centriole is roughly 500 nm long and 300 nm in diameter and the microtubule bundles appear to taper toward the distal end. In addition, we see a handedness to the pericentriolar material at the base (distal end) of the centriole which is opposite to the skew of the microtubule triplets. The region at which the microtubule barrel joins this base is intriguingly complex and includes an internal cylindrical feature which is a site of γ tubulin localization.     

Higher plant microtubule-associated proteins (MAPs): A survey

Summary— Microtubule-associated proteins (MAPs) are one of the factors which regulate the different properties of microtubules during cell cycle and differentiation. They have been characterized as proteins which promote tubulin assembly in a concentration-dependent manner and bind to the outer surface of the polymers in vitro. Most of our knowledge comes from studies of neural microtubule-associated proteins and recent results highlight their implication in neuronal morphogenesis. In contrast, until recently, few data are available about the proteins that associate with plant tubulins. This is due principally to the fact that plant microtubule-associated proteins cannot be purified by the standard procedures used for neural microtubule-associated proteins. First, we will describe methods which have been used to isolate these proteins in plant cells. We will then discuss the biochemical and immunological properties of the plant microtubule-associated proteins which have been isolated. From these results, putative functions can be proposed for these proteins n the particular plant cytoskeleton activities.     

c-Abl kinase-mediated phosphorylation of γ-tubulin promotes γ-tubulin ring complexes assembly and microtubule nucleation

Cytoskeletal microtubules (MTs) are nucleated from γ-tubulin ring complexes (γTuRCs) located at MT organizing centers (MTOCs), such as the centrosome. However, the exact regulatory mechanism of γTuRC assembly is not fully understood. Here, we showed that the nonreceptor tyrosine kinase c-Abl was associated with and phosphorylated γ-tubulin, the essential component of the γTuRC, mainly on the Y443 residue by in vivo (immunofluorescence and immunoprecipitation) or in vitro (surface plasmon resonance) detection. We further demonstrated that phosphorylation deficiency significantly impaired γTuRC assembly, centrosome construction, and MT nucleation. c-Abl/Arg deletion and γ-tubulin Y443F mutation resulted in an abnormal morphology and compromised spindle function during mitosis, eventually causing uneven chromosome segregation. Our findings reveal that γTuRC assembly and nucleation function are regulated by Abl kinase-mediated γ-tubulin phosphorylation, revealing a fundamental mechanism that contributes to the maintenance of MT function.     

Trafficking of cholinesterases and neuroligins mutant proteins. An association with autism

Autism encompasses a wide spectrum of disorders arising during brain development. Recent studies reported that sequence polymorphisms in neuroligin-3 (NLGN3) and neuroligin-4 (NLGN4) genes have been linked to autism spectrum disorders indicating neuroligin genes as candidate targets in brain disorders. We have characterized a single mutation found in two affected brothers that substituted Arg451 to Cys in NL3. Our data show that the exposed Cys causes retention of the protein in the endoplasmic reticulum (ER) when expressed in HEK-293 cells. To examine whether the introduction of a Cys in the C-terminal region of other alpha/beta-hydrolase fold proteins could promote the same cellular phenotype, we made homologous mutations in acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) and found a similar processing deficiency and intracellular retention (De Jaco et al., J Biol Chem. 2006, 281:9667-76). NL3, AChE and BChE mutant proteins are recognized as misfolded in the ER, and degraded via the proteasome pathway. A 2D electrophoresis coupled with mass spectrometry based approach was used to analyze proteins co-immunoprecipitating with NL3 and show differential expression of factors interacting with wild type and mutant NL3. We identified several proteins belonging to distinct ER resident chaperones families, including calnexin, responsible for playing a role in the folding steps of the AChE and NLs.     

Distribution and Characteristics of βII Tubulin-Enriched Microtubules in Interphase Cells

We have used a polyclonal antibody (Ab196) that specifically recognizes the βII tubulin isotype to examine the subcellular distribution and properties of microtubules enriched in this isotype. Antibody specificity was tested by a method that involves the analysis of its interaction with individual β isotypes. Using photoimaging analysis, we observed βII tubulin-enriched microtubules in the perinuclear region, as well as in the microtubules close to the periphery of interphase cells. The observed sorting of βII-enriched microtubules together with the reported increased levels of βII tubulin in taxol-resistant cells (M. Haberet al.,1995,J. Biol. Chem.270, 31269–31275) prompted us to study the behavior of microtubules enriched in this isotype after different depolymerizing treatments. After cold or nocodazol treatments, βII-enriched microtubules anchored at the centrosome and at the cell periphery were observed. In addition, cold-resistant microtubules were marked mainly by the specific anti-βII tubulin antibody but not by anti-acetylated α tubulin, suggesting the presence of different stable microtubule subsets enriched in particular tubulin isoforms.     

Ecdysone oxidase and 3-oxoecdysteroid reductases in Manduca sexta: Developmental changes and tissue distribution

The activities of ecdysone oxidase (EO), 3-oxoecdysteroid 3α-reductase (3α-R), and 3-oxoecdysteroid 3β-reductase (3β-R) were determined for epidermis, hemolymph, and fat body of wandering fifth instar Manduca sexta larvae and for midguts of various developmental stages between 3 days after the last larval and 14 days after the pupal ecdysis. The larval midgut was the only organ showing substantial specific activities of EO and 3α-R, and both increased up to the seventh day after ecdysis. Hemolymph and fat body had only moderate to high 3β-R and low EO activites, and the epidermis did not contain significant activity of any of the enzymes. On the ninth day after the last larval ecdysis the larval midgut epithelium was replaced by a new pupal midgut epithelium. After this event only 3β-R was restored to high activities, whereas EO and 3α-R showed only low to marginal activities. It is concluded that only the larval midgut has a role in the inactivation of ecdysteroids by 3-epimerization. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

DIGESTION IN ADULT FEMALES OF THE LEAF‐FOOTED BUG Leptoglossus zonatus (HEMIPTERA: COREIDAE) WITH EMPHASIS ON THE GLYCOSIDE HYDROLASES α‐AMYLASE, α‐GALACTOSIDASE,AND α‐GLUCOSIDASE

The leaffooted bug, Leptoglossus zonatus (Hemiptera: Coreidae) is an emerging pest of several crops around the World and up to now very little is known of its digestive system. In this article, glycoside hydrolase (carbohydrase) activities in the adult midgut cells and in the luminal contents of L. zonatus adult females were studied. The results showed the distribution of digestive carbohydrases in adults of this heteropteran species in the different intestinal compartments. Determination of the spatial distribution of α‐glucosidase activity in L. zonatus midgut showed only one major molecular form, which was not equally distributed between soluble and membrane‐bound isoforms, being more abundant as a membrane‐bound enzyme. The majority of digestive carbohydrases were found in the soluble fractions. Activities against starch, maltose and the synthetic substrate NPαGlu were found to show the highest levels of activity, followed by enzymes active against galactosyl oligosaccharides. Based on ion‐exchange chromatography elution profiles and banding patterns in mildly denaturing electrophoresis, both midgut α‐amylases and α‐galactosidases showed at least two isoforms. The data suggested that the majority of carbohydrases involved in initial digestion were present in the midgut lumen, whereas final digestion of starch and of galactosyl oligosaccharides takes place partially within the lumen and partially at the cell surface. The complex of carbohydrases here described was qualitatively appropriate for the digestion of free oligosaccharides and oligomaltodextrins released by α‐amylases acting on maize seed starch granules.     

Regulation of Sucrose Metabolism in Higher Plants: Localization and Regulation of Activity of Key Enzymes

ABSTRACT

Sucrose (Sue) plays a central role in plant growth and development. It is a major end product of photosynthesis and functions as a primary transport sugar and in some cases as a direct or indirect regulator of gene expression. Research during the last 2 decades has identified the pathways involved and which enzymes contribute to the control of flux. Availability of metabolites for Sue synthesis and 'demand' for products of sucrose degradation are important factors, but this review specifically focuses on the biosynthetic enzyme sucrose-phosphate synthase (SPS), and the degradative enzymes, sucrose synthase (SuSy), and the invertases. Recent progress has included the cloning of genes encoding these enzymes and the elucidation of posttranslational regulatory mechanisms. Protein phosphorylation is emerging as an important mechanism controlling SPS activity in response to various environmental and endogenous signals. In terms of Sue degradation, invertase-catalyzed hydrolysis generally has been associated with cell expansion, whereas SuSy-catalyzed metabolism has been linked with biosynthetic processes (e.g., cell wall or storage products). Recent results indicate that SuSy may be localized in multiple cellular compartments: (1) as a soluble enzyme in the cytosol (as traditionally assumed); (2) associated with the plasma membrane; and (3) associated with the actin cytoskeleton. Phosphorylation of SuSy has been shown to occur and may be one of the factors controlling localization of the enzyme. The purpose of this review is to summarize some of the recent developments relating to regulation of activity and localization of key enzymes involved in sucrose metabolism in plants.     

Regulation of tubulin synthesis and cell cycle progression in mammalian cells by gamma-tubulin-mediated microtubule nucleation

We have previously shown that gamma-tubulin, the third member of the tubulin family that functions in microtubule nucleation, when overexpressed, accumulates throughout the cytoplasm and forms numerous ectopic microtubule nucleation sites in mammalian cells (Shu and Joshi [1995] J. Cell. Biol. 130:1137-1147). We now show that overexpression of gamma-tubulin differentially upregulates the synthesis of alpha- and beta-tubulins in mammalian cells. Surprisingly, despite a dramatic increase in the level of gamma-tubulin protein in transfected cells, there is no obvious alteration in the level of endogenous gamma-tubulin mRNA, suggesting that synthesis of gamma-tubulin might employ a regulatory mechanism other than the autoregulatory pathway shared by alpha- and beta-tubulins. Interestingly, a significant number of mammalian cells transfected with gamma-tubulin fail to form normal bipolar mitotic spindle during mitosis; instead, numerous microtubules occur in the cytoplasm intermingled with the condensed chromosomes. In addition, they reduplicate their DNA after an abnormal mitotic exit. These results thus suggest that the number of microtubule nucleation sites, or even gamma-tubulin itself, might play an important role in the regulation of tubulin synthesis as well as cell cycle progression.     

Regulation of Sucrose Metabolism in Higher Plants: Localization and regulation of Activity of Key Enzymes

Sucrose (Suc) plays a central role in plant growth and development. It is a major end product of photosynthesis and functions as a primary transport sugar and in some cases as a direct or indirect regulator of gene expression. Research during the last 2 decades has identified the pathways involved and which enzymes contribute to the control of flux. Availability of metabolites for Suc synthesis and ‘demand’ for products of sucrose degradation are important factors, but this review specifically focuses on the biosynthetic enzyme sucrose-phosphate synthase (SPS), and the degradative enzymes, sucrose synthase (SuSy), and the invertases. Recent progress has included the cloning of genes encoding these enzymes and the elucidation of posttranslational regulatory mechanisms. Protein phosphorylation is emerging as an important mechanism controlling SPS activity in response to various environmental and endogenous signals. In terms of Suc degradation, invertase-catalyzed hydrolysis generally has been associated with cell expansion, whereas SuSy-catalyzed metabolism has been linked with biosynthetic processes (e.g., cell wall or storage products). Recent results indicate that SuSy may be localized in multiple cellular compartments: (1) as a soluble enzyme in the cytosol (as traditionally assumed); (2) associated with the plasma membrane; and (3) associated with the actin cytoskel-eton. Phosphorylation of SuSy has been shown to occur and may be one of the factors controlling localization of the enzyme. The purpose of this review is to summarize some of the recent developments relating to regulation of activity and localization of key enzymes involved in sucrose metabolism in plants.     

Map7/7D1 and Dvl form a feedback loop that facilitates microtubule remodeling and Wnt5a signaling

The Wnt signaling pathway can be grouped into two classes, the β‐catenin‐dependent and β‐catenin‐independent pathways. Wnt5a signaling through a β‐catenin‐independent pathway promotes microtubule (MT) remodeling during cell‐substrate adhesion, cell migration, and planar cell polarity formation. Although Wnt5a signaling and MT remodeling are known to form an interdependent regulatory loop, the underlying mechanism remains unknown. Here we show that in HeLa cells, the paralogous MT‐associated proteins Map7 and Map7D1 (Map7/7D1) form an interdependent regulatory loop with Disheveled, the critical signal transducer in Wnt signaling. Map7/7D1 bind to Disheveled, direct its cortical localization, and facilitate the cortical targeting of MT plus‐ends in response to Wnt5a signaling. Wnt5a signaling also promotes Map7/7D1 movement toward MT plus‐ends, and depletion of the Kinesin‐1 member Kif5b abolishes the Map7/7D1 dynamics and Disheveled localization. Furthermore, Disheveled stabilizes Map7/7D1. Intriguingly, Map7/7D1 and its Drosophila ortholog, Ensconsin show planar‐polarized distribution in both mouse and fly epithelia, and Ensconsin influences proper localization of Drosophila Disheveled in pupal wing cells. These results suggest that the role of Map7/7D1/Ensconsin in Disheveled localization is evolutionarily conserved.     

A novel approach to predicting protein structural classes in a (20–1)-D amino acid composition space

The development of prediction methods based on statistical theory generally consists of two parts: one is focused on the exploration of new algorithms, and the other on the improvement of a training database. The current study is devoted to improving the prediction of protein structural classes from both of the two aspects. To explore a new algorithm, a method has been developed that makes allowance for taking into account the coupling effect among different amino acid components of a protein by a covariance matrix. To improve the training database, the selection of proteins is carried out so that they have (1) as many non-homologous structures as possible, and (2) a good quality of structure. Thus, 129 representative proteins are selected. They are classified into 30 α, 30 β, 30 α + β, 30 α/β, and 9 ζ (irregular) proteins according to a new criterion that better reflects the feature of the structural classes concerned. The average accuracy of prediction by the current method for the 4 × 30 regular proteins is 99.2%, and that for 64 independent testing proteins not included in the training database is 95.3%. To further validate its efficiency, a jackknife analysis has been performed for the current method as well as the previous ones, and the results are also much in favor of the current method. To complete the mathematical basis, a theorem is presented and proved in Appendix A that is instructive for understanding the novel method at a deeper level. © 1995 Wiley-Liss, Inc.