Anne Roobol

Evidence that griseofulvin binds to a microtubule associated protein.

We have recently shown that the anti-mitotic drug, griseofulvin, inhibits assembly of brain microtubules in vitro [l] . Similar data have been presented by Weber et al. [2] . Our earlier experiments indicated that griseofulvin inhibits microtubule assembly by preventing the association between tubulin and the microtubule associated proteins (MAPS) which normally co-purify with tubulin [3] and promote assembly of purified tubulin dimer [4] . We now present evidence that griseofulvin binds specifically to MAPS and not to tubulin dimer at concentrations of griseofulvin which inhibits microtubule assembly.

Biochemical insights into the mechanisms central to the response of mammalian cells to cold stress and subsequent rewarming

Mammalian cells cultured in vitro are able to recover from cold stress. However, the mechanisms activated during cold stress and recovery are still being determined. We here report the effects of hypothermia on cellular architecture, cell cycle progression, mRNA stability, protein synthesis and degradation in three mammalian cell lines. The cellular structures examined were, in general, well maintained during mild hypothermia (27–32 °C) but became increasingly disrupted at low temperatures (4–10 °C). The degradation rates of all mRNAs and proteins examined were much reduced at 27 °C, and overall protein synthesis rates were gradually reduced with temperature down to 20 °C. Proteins involved in a range of cellular activities were either upregulated or downregulated at 32 and 27 °C during cold stress and recovery. Many of these proteins were molecular chaperones, but they did not include the inducible heat shock protein Hsp72. Further detailed investigation of specific proteins revealed that the responses to cold stress and recovery are at least partially controlled by modulation of p53, Grp75 and eIF3i levels. Furthermore, under conditions of severe cold stress (4 °C), lipid‐containing structures were observed that appeared to be in the process of being secreted from the cell that were not observed at less severe cold stress temperatures. Our findings shed light on the mechanisms involved and activated in mammalian cells upon cold stress and recovery.

A comparison of tubulins from mammalian brain and Physarumpolycephalum using SDS—polyacrylamide gel electrophoresis and peptide mapping

IBM isolated from rn~rn~~ brain consists of two equimdar protein species designated (Y- and /?-tubulins. Conventionally cw-tubulin is the slower, and P-tubulin the faster migrating species during sodium dodecyl sulphate-polyacrylamide gel electrophoresis [ 11. Tubulin has been considered to be a highly conserved protein ]2,3]; however, we now show differences between tubulins from mammalian brain and myxamoebae of the slime mould Physarum poZycephaZum from which microtubule proteins have been purified by an assembly procedure [4]. Evidence is baaed on differences in migration of these tubulins on SDS- and urea-SDS-PAGE, and peptide mapping, and demonstrates that Physarum tubulin consists of an ol-tubulin which is similar to brain /3-tubulin, and a P-tubulin which is dissimilar to both brain subunits. In addition, the analogous proteins from Physarum and brain demonstrate micro heterogeneity during urea-SDS-PAGE. The migration of the Physarum P-tubulin is altered during urea-SDS-PAGE such as to cause the order of migration of the myxamoebal tubulins to be reversed with respect to that during SDS-PAGE. In addition the importance of the source of SDS in resolving tubu~ subunit proteins during PAGE is highlighted. Abbreviations: EGTA, ethylene glycol-bis@aminoethyl ether) tetra acetic acid; PAGE, polyac~~rn~e gel electrophoresis; PfPES,pi~r~~e-~~~is(2~th~e~~pho~c acid); SDS, sodium dodecyl sulphate

In vitro assembly of microtubule proteins from myxamoebae of Physarum polycephalum

Abstract Microtubule protein of >95% purity has been isolated by self-assembly from concentrated cell extracts of myxamoebae of Physarum polycephalum . Ninety-eight percent of the amoebal microtubule protein was tubulin. Both a and β subunits of amoebal tubulin were different from neurotubulin α and β subunits, but very similar to those of Tetrahymena ciliary tubulin. The non-tubulin components, which co-purified with tubulin through three assembly cycles, were essential to microtubule formation and contained several polypeptides including some of apparent molecular weights 49000, 57000 and 59000. Purified amoebal microtubule protein formed microtubules on warming in the absence of glycerol which were cold- and Ca 2+ -labile. In vitro, microtubule assembly was inhibited by vinblastine, benzimidazole derivatives and griseofulvin, but not by 10 −4 M colchicine. Amoebal tubulin had a much lower affinity than neurotubulin for colchicine.

Inhibition by griseofulvin of microtubule assembly in vitro

The antimitotic drug, griseofulvin, has been shown to arrest cell division at a point close to metaphase in a variety of cells [1]. The microtubules in griseofulvinblocked HeLa cells appear to have a normal morphology and orientation within the spindle [2] and it has been suggested that griseofulvin inhibits some aspect of microtubule function rather than microtubule assembly [3]. Using a crude extract of brain, Wilson and Bryan [1 ] were unable to inhibit in vitro microtubule assembly, as measured by electron microscopy, with griseofulvin at a concentration known to inhibit mitosis (60/aM). We report here that griseofulvin at concentration between 20 and 200/IM does inhibit in vitro assembly of purified microtubule protein from brain as measured by turbidimetry, electron microscopy and a sedimentation method. Inhibition of assembly by griseofulvin is distinctly different from inhibition by colchicine. Disc gel electrophoretic analysis indicates that microtubule protein polymerized in the presence of griseofulvin is depleted in the high molecular weight (HMW) components which normally copurify with tubulin [4,5].

Eukaryotic chaperonin containing T-complex polypeptide 1 interacts with filamentous actin and reduces the initial rate of actin polymerization in vitro

Abstract We have previously observed that subunits of the chaperonin required for actin production (type-II chaperonin containing T-complex polypeptide 1 [CCT]) localize at sites of microfilament assembly. In this article we extend this observation by showing that substantially substoichiometric CCT reduces the initial rate of pyrene-labeled actin polymerization in vitro where eubacterial chaperonin GroEL had no such effect. CCT subunits bound selectively to F-actin in cosedimentation assays, and CCT reduced elongation rates from both purified actin filament “seeds” and the short and stabilized, minus-end blocked filaments in erythrocyte membrane cytoskeletons. These observations suggest CCT might remain involved in biogenesis of the actin cytoskeleton, by acting at filament (+) ends, beyond its already well-established role in producing new actin monomers.

The Cotranslational Contacts between Ribosome-bound Nascent Polypeptides and the Subunits of the Hetero-oligomeric Chaperonin TRiC Probed by Photocross-linking

The hetero-oligomeric eukaryotic chaperonin TRiC (TCP-1-ring complex, also called CCT) interacts cotranslationally with a diverse subset of newly synthesized proteins, including actin, tubulin, and luciferase, and facilitates their correct folding. A photocross-linking approach has been used to map the contacts between individual chaperonin subunits and ribosome-bound nascent chains of increasing length. Whereas a cryo-EM study suggests that chemically denatured actin interacts with only two TRiC subunits (δ and either β or ϵ), actin and luciferase chains photocross-link to at least six TRiC subunits (α, β, δ, ϵ, ξ, and θ) at different stages of translation. Furthermore, the photocross-linking of actin, but not luciferase, nascent chains to TRiC subunits ζ and θ was length-dependent. In addition, a single photoreactive probe incorporated at a unique site in actin nascent chains of different lengths reacted covalently with multiple TRiC subunits, thereby indicating that the nascent chain samples the polypeptide binding sites of different subunits. We conclude that elongating actin and luciferase nascent chains contact multiple TRiC subunits upon emerging from the ribosome, and that the TRiC subunits contacted by nascent actin change as it elongates and starts to fold.

Slow axonal transport of the cytosolic chaperonin CCT with Hsc73 and actin in motor neurons

Molecular chaperones are well known for their role in facilitating the folding of nascent and newly synthesized proteins, but have other roles, including the assembly, translocation and renaturation of intracellular proteins. Axons are convenient tissues for the study of some of these other roles because they lack the capacity for significant protein synthesis. We examine the axonal transport of the cytosolic chaperonin containing T‐ complex polypeptide 1 (CCT) by labeling lumbar motor neurons with [35S]methionine and examining sciatic nerve proteins by 2‐D gel electrophoresis and immunoblotting. All CCT subunits identifiable with specific antibodies, namely CCTα, CCTβ, CCTγ and CCTϵ/CCTθ (the latter two subunits colocalized in analyses of rat nerve samples), appeared to be labeled in “slow component b” of axonal transport along with the molecular chaperone Hsc73 and actin, a major folding substrate for CCT. Our results are consistent with molecular chaperones having a post‐translational role in maintaining the native form of actin during its slow transport to the axon terminal and ensuring its correct assembly into microfilaments.

Post-Translational Events of a Model Reporter Protein Proceed With Higher Fidelity and Accuracy Upon Mild Hypothermic Culturing of Chinese Hamster Ovary Cells

Chinese hamster ovary cells (CHO) are routinely used in industry to produce recombinant therapeutic proteins and a number of studies have reported increased recombinant mRNA levels at temperatures <37 degrees C. Surprisingly, the effect of reduced temperature on mRNA translation in CHO cells has not been investigated despite this process being highly responsive to environmental stresses. The relationship between low temperature culturing of CHO cells and mRNA translation was therefore investigated using labeling studies and dual luciferase reporter gene technology. Global protein synthetic capacity was not greatly affected at 32 degrees C but was diminished at lower temperatures. The expression of both cap-dependent and cap-independent (IRES driven) mRNA translated luciferase reporter gene activity was highest at 32 degrees C on a per cell basis and this was partially accounted for by increased mRNA levels. Importantly, post-translational events appear to proceed with higher fidelity and accuracy at 32 than 37 degrees C resulting in increased yield of active protein as opposed to an increase in total polypeptide synthesis. Therefore at 32 degrees C recombinant cap-dependent mRNA translation appears sufficient to maintain recombinant protein yields on a per cell basis and this is associated with improved post-translational processing.

Disassembly of the Cytosolic Chaperonin in Mammalian Cell Extracts at Intracellular Levels of K+ and ATP

The eukaryotic, cytoplasmic chaperonin, CCT, is essential for the biogenesis of actin- and tubulin-based cytoskeletal structures. CCT purifies as a doubly toroidal particle containing two eight-membered rings of ∼60-kDa ATPase subunits, each encoded by an essential and highly conserved gene. However, immunofluorescence detection with subunit-specific antibodies has indicated that in cells CCT subunits do not always co-localize. We report here that CCT ATPase activity is highly dependent on K+ ion concentration and that in cell extracts, at physiological levels of K+and ATP, there is considerable dissociation of CCT to a smaller oligomeric structure and free subunits. This dissociation is consequent to ATP hydrolysis and is readily reversed on removal of ATP. The ranking order for ease with which subunits can exit the chaperonin particle correlates well with the length of a loop structure, identified by homology modeling, in the intermediate domain of CCT subunits. K+-ATP-induced disassembly is not an intrinsic property of purified CCT over a 40-fold concentration range and requires the presence of additional factor(s) present in cell extracts.