Tumors with nonfunctional retinoblastoma protein are killed by reduced γ-tubulin levels

In various tumors inactivation of growth control is achieved by interfering with the RB1 signaling pathway. Here, we describe that RB1 and γ-tubulin proteins moderate each other's expression by binding to their respective gene promoters. Simultaneous reduction of RB1 and γ-tubulin protein levels results in an E2F1-dependent up-regulation of apoptotic genes such as caspase 3. We report that in various tumors types, there is an inverse correlation between the expression levels of γ-tubulin and RB1 and that in tumor cell lines with a nonfunctioning RB1, reduction of γ-tubulin protein levels leads to induction of apoptosis. Thus, the RB1/γ-tubulin signal network can be considered as a new target for cancer treatment.     

Influences of the interference of γ-tubulin gene expression on the morphology and microtubules of ciliate Euplotes eurystomus

A recombinant plasmid L4440-GTU expressing γ-tubulin dsRNA of the ciliate Euplotes eurystomus was constructed and transferred into Escherichia coli strain HT115. The resultant E. coli bacteria were fed on E. eurystomus to inhibit the ciliate's γ-tubulin gene expression. As a result, the γ-tubulin gene expression level was decreased, and this inactivation blocked cell division, which was lethal. In addition, the loss of the C-tubule from the nine-microtubule triplets in basal bodies, and the disappearance of some microtubules or mislocalization of some microtubule organization units in the subpellicular microtubule layers were also observed. These results indicate that the γ-tubulin is not only important for the stability of the nine-microtubule triplets in basal bodies, but also necessary for the integrity of microtubule organization patterns in the subpellicular microtubule layer.     

Polar organizers and girdling bands of microtubules are associated with γ-tubulin and act in establishment of meiotic quadripolarity in the hepatic Aneura pinguis (Bryophyta)

Summary. Meiosis in Aneura pinguis is preceded by extensive cytoplasmic preparation for quadripartitioning of the diploid sporocyte into a tetrad of haploid spores. In early prophase the four future spore domains are defined by lobing of the cytoplasm and development of a quadripolar prophase spindle focused at polar organizers (POs) centered in the lobes. Cells entering the reproductive phase become isolated and, instead of hooplike cortical microtubules, have endoplasmic microtubule systems centered on POs. These archesporial cells proliferate by mitosis before entering meiosis. In prophase of each mitosis, POs containing a distinct concentration of γ-tubulin appear de novo at tips of nuclei and initiate the bipolar spindle. Cells entering meiosis become transformed into quadrilobed sporocytes with four POs, one in each lobe. This transition is a complex process encompassing assembly of two opposite POs which subsequently disperse into intersecting bands of microtubules that form around the central nucleus. The girdling bands define the future planes of cytokinesis and the cytoplasm protrudes through the restrictive bands becoming quadrilobed. Two large POs reappear in opposite cleavage furrows. Each divides and the resulting POs migrate into the tetrahedral lobes of cytoplasm. Cones of microtubules emanating from the four POs interact to form a quadripolar microtubule system (QMS) that surrounds the nucleus in meiotic prophase. The QMS is subsequently transformed into a functionally bipolar metaphase spindle by migration of poles in pairs to opposite cleavage furrows. These findings contribute to knowledge of microtubule organization and the role of microtubules in spatial regulation of cytokinesis in plants. Correspondence and reprints: Department of Biology, University of Louisiana-Lafayette, Lafayette, LA 70504-2451, U.S.A.     

Kinesin-14 Pkl1 targets γ-tubulin for release from the γ-tubulin ring complex (γ-TuRC) ‬‬‬‬‬‬‬

The γ-tubulin ring complex (γ-TuRC) is a key part of microtubule-organizing centers (MTOCs) that control microtubule polarity, organization and dynamics in eukaryotes. Understanding regulatory mechanisms of γ-TuRC function is of fundamental importance, as this complex is central to many cellular processes, including chromosome segregation, fertility, neural development, T-cell cytotoxicity and respiration. The fission yeast microtubule motor kinesin-14 Pkl1 regulates mitosis by binding to the γ-tubulin small complex (γ-TuSC), a subunit of γ-TuRC. Here we investigate the binding mechanism of Pkl1 to γ-TuSC and its functional consequences using genetics, biochemistry, peptide assays and cell biology approaches in vivo and in vitro. We identify two critical elements in the Tail domain of Pkl1 that mediate γ-TuSC binding and trigger release of γ-tubulin from γ-TuRC. Such action disrupts the MTOC and results in failed mitotic spindle assembly. This study is the first demonstration that a motor protein directly affects the structural composition of the γ-TuRC, and we provide details of this mechanism that may be of broad biological importance.     

NME7 is a functional component of the γ-tubulin ring complex

As the primary microtubule nucleator in animal cells, the γ-tubulin ring complex (γTuRC) plays a crucial role in microtubule organization, but little is known about how the activity of the γTuRC is regulated. Recently, isolated γTuRC was found to contain NME7, a poorly characterized member of the NME family. Here we report that NME7 is a γTuRC component that regulates the microtubule-nucleating activity of the γTuRC. NME7 contains two putative kinase domains, A and B, and shows autophosphorylating activity. Whereas domain A is involved in the autophosphorylation, domain B is inactive. NME7 interacts with the γTuRC through both A and B domains, with Arg-322 in domain B being crucial to the binding. In association with the γTuRC, NME7 localizes to centrosomes throughout the cell cycle and to mitotic spindles during mitosis. Suppression of NME7 expression does not affect γTuRC assembly or localization to centrosomes, but it does impair centrosome-based microtubule nucleation. Of importance, wild-type NME7 promotes γTuRC-dependent nucleation of microtubules, but kinase-deficient NME7 does so only poorly. These results suggest that NME7 functions in the γTuRC in a kinase-dependent manner to facilitate microtubule nucleation.     

Accumulation of maize γ-zein and γ-zein: KDEL to high levels in tobacco leaves and differential increase of BiP synthesis in transformants

Two gene constructs (pROK.TG1L and pROK.TG1LK) were utilized to achieve accumulation of maize γ-zein to high levels in tobacco (Nicotiana tabacum L.) leaves. Both the chimaeric genes contained the γ-zein-coding region preceded by the 5′untranslated leader from the coat protein mRNA of TMV, but one of them (pROK.TG1LK) was modified in its protein-coding region by the addition of the ER retention signal KDEL. The accumulation of γ-zein and γ-zein:KDEL in leaves was compared with heterologous protein accumulation in tobacco plants previously transformed with a γ-zein cDNA harbouring a native 5′UTR. Replacement of γ-zein 5′UTR with the TMV leader dramatically increased γ-zein production. Furthermore, γ-zein:KDEL-expressing plants, on average, accumulated twice as much foreign protein in their leaves as pROK.TG1L plants. The two-fold increase in the level of γ-zein:KDEL can probably be attributed to an improvement in the mechanism for ER retention of zeins in the transgenic cells. Transformants also showed increased production of BiP, though to a lesser extent in γ-zein:KDEL-expressing plants compared with pROK.TG1L plants. It is therefore likely that γ-zein:KDEL retention is made less dependent on the chaperone assistance of BiP by the presence of the KDEL signal on the γ-zein mutant. Received: 15 October 1999 / Accepted: 28 February 2000     

Relationship between the Golgi complex and microtubules enriched in detyrosinated or acetylated α-tubulin: studies on cells recovering from nocodazole and cells in the terminal phase of cytokinesis

Double immunofluorescence microscopy was used to study the relationship between the Golgi complex and microtubules enriched in posttranslationally modified tubulins in cultured mouse L929 fibroblasts. In interphase cells, the elements of the Golgi complex were grouped around the microtubule-organizing center. From here, tyrosinated microtubules extended to the periphery of the cells, whereas the distribution of detyrosinated and acetylated microtubules largely overlapped with that of the Golgi complex. Treatment of cells with 10 M nocodazole led to the disruption of all microtubules and dispersion of the Golgi elements. Following withdrawal of the drug, tyrosinated microtubules reformed first, followed by acetylated and then detyrosinated microtubules. In parallel, the Golgi elements moved back toward the juxtanuclear region and reestablished a close spatial relationship first with the acetylated and later also with the detyrosinated microtubules. Long-term recovery in the presence of 0.15 or 0.3 M nocodazole allowed partial reformation of tyrosinated and acetylated microtubules, whereas no or only a few detyrosinated microtubules were detected. At the same time, the Golgi elements were grouped closer together around or on one side of the nucleus in close relation to acetylated microtubules. In synchronized cells released from a mitotic block, a radiating array of tyrosinated microtubules was first formed, followed by acetylated and detyrosinated microtubules. The Golgi elements initially came together in a few groups and thereafter took an overall morphology similar to that in interphase cells. During this reunification, they showed a close spatial relationship to acetylated microtubules, whereas detyrosinated microtubules appeared only later. Microtubules enriched in acetylated and/or detyrosinated tubulin thus appear to take part in establishing and maintaining the organization of the Golgi elements within an interconnected supraorganellar system. Whether the acetylation and detyrosination of tubulin are directly involved in this process or merely represent two modifications within this subpopulation of microtubules remains unknown.On leave of absence from the Department of Histology and Embryology, Institute of Biostructure, Medical School, Warsaw, Poland     

Regulation of seedling growth by ethylene and the ethylene–auxin crosstalk

Planta - This review highlights that the auxin gradient, established by local auxin biosynthesis and transport, can be controlled by ethylene, and steers seedling growth. A better understanding of...     

The length and disposition of cortical microtubules in plant cells fixed in glutaraldehyde—Osmium tetroxide

Serial sectioning has been used to show that the majority of circumferential microtubules lying in the cortex of root tip cells are much shorter than the cell circumference. The significance of this observation is briefly discussed.     

Mutations in the TGFβ binding-protein-like domain 5 of FBN1 are responsible for acromicric and geleophysic dysplasias

Geleophysic (GD) and acromicric dysplasia (AD) belong to the acromelic dysplasia group and are both characterized by severe short stature, short extremities, and stiff joints. Although AD has an unknown molecular basis, we have previously identified ADAMTSL2 mutations in a subset of GD patients. After exome sequencing in GD and AD cases, we selected fibrillin 1 (FBN1) as a candidate gene, even though mutations in this gene have been described in Marfan syndrome, which is characterized by tall stature and arachnodactyly. We identified 16 heterozygous FBN1 mutations that are all located in exons 41 and 42 and encode TGFβ-binding protein-like domain 5 (TB5) of FBN1 in 29 GD and AD cases. Microfibrillar network disorganization and enhanced TGFβ signaling were consistent features in GD and AD fibroblasts. Importantly, a direct interaction between ADAMTSL2 and FBN1 was demonstrated, suggesting a disruption of this interaction as the underlying mechanism of GD and AD phenotypes. Although enhanced TGFβ signaling caused by FBN1 mutations can trigger either Marfan syndrome or GD and AD, our findings support the fact that TB5 mutations in FBN1 are responsible for short stature phenotypes.     

Evidence for three “classes” of microtubules in the interpolar space of the mitotic micronucleus of a ciliate and the participation of the nuclear envelope in conferring stability to microtubules

Exposure of Nyctotherus ovalis to low temperatures or vinblastine caused similar reactions of classes of microtubules (mt) present in the mitotic micronucleus of this ciliate towards both treatments. However, differences of sensitivity between certain classes of mt at individual mitotic stages exist. Unlike the kinetochore mt (kmt) of most other eukaryotic cells, kmt in Nyctotherus completely disassemble after incubation at 6–8 ° C (60 min) and most disappear after prolonged exposure to vinblastine (10^–5 M, 16 h). The depolymerization of kmt causes the collapse of the spindle and a dislocation of chromosomes at metaphase, yet the reduced number of kmt after vinblastine-treatment still allows an alignment of composite complexes at the spindle equator. The data suggest that three individual sets of mt exist in the interpolar spindle region during ana- and telophase: 1) interpolar mt (int mt), which are assembled during anaphase, are cold- and vinblastine sensitive; 2) manchette mt (ma mt), which are first observed underneath the nuclear envelope during mid-anaphase, are cold-stable and insensitive to vinblastine treatment (10^–5 M); after prolonged treatment (16 h) they form spiral structures; 3) stembody mt (st mt), comprising the interpolar region of the nucleus during telophase, are cold- and vinblastine insensitive. Paracrystalline structures resembling a stembody are formed in telophase-like division stages after prolonged vinblastine exposure (16 h, 10^–5 M). Since kmt and int mt possess the same sensitivity under depolymerizing conditions, they probably have a similar composition. Thus the idea that the int mt in this organism arise by elongation of kmt is supported. However, st mt apparently do not originate from an extension of preexistent int mt, but appear to represent a new set of stable mt. This is emphasized not only by their greater stability compared to the int mt but also by the distribution of cold-stable mt in late anaphase micronuclei. The ma mt may be an intermediary step in formation of st mt since their stability resembles that of the st mt. A comparison of the substructure of vinblastine-induced paracrystals in Nyctotherus with those observed in in vitro systems with known composition suggests that a turnover of MAPs may be responsible for the different stability of mt and thus could specify and regulate mt sensitivity and function. Another organelle, possibly involved in conferring stability to mt, is the nuclear membrane. The assumption that the nuclear envelope possesses an intrinsic property to nucleate mt and thus aid in the alignment of mt is supported.     

Structure and mechanism of the γ-secretase intramembrane protease complex

γ-Secretase is a membrane protein complex that proteolyzes within the transmembrane domain of >100 substrates, including those derived from the amyloid precursor protein and the Notch family of cell surface receptors. The nine-transmembrane presenilin is the catalytic component of this aspartyl protease complex that carries out hydrolysis in the lipid bilayer. Advances in cryoelectron microscopy have led to the elucidation of the structure of the γ-secretase complex at atomic resolution. Recently, structures of the enzyme have been determined with bound APP- or Notch-derived substrates, providing insight into the nature of substrate recognition and processing. Molecular dynamics simulations of substrate-bound enzymes suggest dynamic mechanisms of intramembrane proteolysis. Structures of the enzyme bound to small-molecule inhibitors and modulators have also been solved, setting the stage for rational structure-based drug discovery targeting γ-secretase.     

Carbendazim-resistance associated β2-tubulin substitutions increase deoxynivalenol biosynthesis by reducing the interaction between β2-tubulin and IDH3 in Fusarium graminearum

Microtubule is a well-known structural protein participating in cell division, motility and vesicle traffic. In this study, we found that β₂-tubulin, one of the microtubule components, plays an important role in regulating secondary metabolite deoxynivalenol (DON) biosynthesis in Fusarium graminearum by interacting with isocitrate dehydrogenase subunit 3 (IDH3). We found IDH3 negatively regulate DON biosynthesis by reducing acetyl-CoA accumulation in F. graminearum and DON biosynthesis was stimulated by exogenous acetyl-CoA. In addition, the expression of IDH3 significantly decreased in the carbendazim-resistant mutant nt167 (Fgβ₂^F167Y). Furthermore, we found that carbendazim-resistance associated β₂-tubulin substitutions reducing the interaction intensity between β₂-tubulin and IDH3. Interestingly, we demonstrated that β₂-tubulin inhibitor carbendazim can disrupt the interaction between β₂-tubulin and IDH3. The decreased interaction intensity between β₂-tubulin and IDH3 resulted in the decreased expression of IDH3, which can cause the accumulation of acetyl-CoA, precursor of DON biosynthesis in F. graminearum. Thus, we revealed that carbendazim-resistance associated β₂-tubulin substitutions or carbendazim treatment increases DON biosynthesis by reducing the interaction between β₂-tubulin and IDH3 in F. graminearum. Taken together, the novel findings give the new perspectives of β₂-tubulin in regulating secondary metabolism in phytopathogenic fungi.     

Inhibition of gluconeogenesis in the kidney cortex by γ-hydroxyglutamate and γ-hydroxy-α-ketoglutarate

γ-Hydroxyglutamate, γ-hydroxy-α-ketoglutarate and glyoxylate inhibit kidney gluconeogenesis from pyruvate and lactate strongly but have little effect on glucose synthesis from Krebs cycle intermediates. The inhibitory effect in the presence of pyruvate appears to result from a block in the Krebs cycle between citrate and α-ketoglutarate.     

The GCP3-interacting proteins GIP1 and GIP2 are required for γ-tubulin complex protein localization, spindle integrity, and chromosomal stability

Microtubules (MTs) are crucial for both the establishment of cellular polarity and the progression of all mitotic phases leading to karyokinesis and cytokinesis. MT organization and spindle formation rely on the activity of γ-tubulin and associated proteins throughout the cell cycle. To date, the molecular mechanisms modulating γ-tubulin complex location remain largely unknown. In this work, two Arabidopsis thaliana proteins interacting with gamma-tubulin complex protein3 (GCP3), GCP3-interacting protein1 (GIP1) and GIP2, have been characterized. Both GIP genes are ubiquitously expressed in all tissues analyzed. Immunolocalization studies combined with the expression of GIP-green fluorescent protein fusions have shown that GIPs colocalize with γ-tubulin, GCP3, and/or GCP4 and reorganize from the nucleus to the prospindle and the preprophase band in late G2. After nuclear envelope breakdown, they localize on spindle and phragmoplast MTs and on the reforming nuclear envelope of daughter cells. The gip1 gip2 double mutants exhibit severe growth defects and sterility. At the cellular level, they are characterized by MT misorganization and abnormal spindle polarity, resulting in ploidy defects. Altogether, our data show that during mitosis GIPs play a role in γ-tubulin complex localization, spindle stability and chromosomal segregation.     

The very many faces of presenilins and the γ-secretase complex

Presenilin is a central, catalytic component of the γ-secretase complex which conducts intramembrane cleavage of various protein substrates. Although identified and mainly studied through its role in the development of amyloid plaques in Alzheimer disease, γ-secretase has many other important functions. The complex seems to be evolutionary conserved throughout the Metazoa, but recent findings in plants and Dictyostelium discoideum as well as in archeons suggest that its evolution and functions might be much more diversified than previously expected. In this review, a selective survey of the multitude of functions of presenilins and the γ-secretase complex is presented. Following a brief overview of γ-secretase structure, assembly and maturation, three functional aspects are analyzed: (1) the role of γ-secretase in autophagy and phagocytosis; (2) involvement of the complex in signaling related to endocytosis; and (3) control of calcium fluxes by presenilins.     

Prostaglandins act as neurotoxin for differentiated neuroblastoma cells in culture and increase levels of ubiquitin and β-amyloid

Summary Although chronic inflammatory reactions have been proposed to cause neuronal degeneration associated with Alzheimer’s disease (AD), the role of prostaglandins (PGs), one of the secretory products of inflammatory reactions, in degeneration of nerve cells has not been studied. Our initial observation that PGE₁-induced differentiated neuroblastoma (NB) cells degenerate in vitro more rapidly than those induced by RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, has led us to postulate that PGs act as a neurotoxin. This study has further investigated the effects of PGs on differentiated NB cells in culture. Results showed that PGA₁ was more effective than PGE₁ in causing degeneration of differentiated NB cells as shown by the cytoplasmic vacuolation and fragmentation of soma, nuclei, and neurites. Because increased levels of ubiquitin and β-amyloid have been implicated in causing neuronal degeneration, we studied the effects of PGs on the levels of these proteins during degeneration of NB cells in vitro by an immunostaining technique, using primary antibodies to ubiquitin and β-amyloid. Results showed that PGs increased the intracellular levels of ubiquitin and β-amyloid prior to degeneration, whereas the degenerated NB cells had negligible levels of these proteins. These data suggest that PGs act as external neurotoxic signals which increase levels of ubiquitin and β-amyloid that represent one of the intracellular signals for initiating degeneration of nerve cells.