Enolase is present at the centrosome of HeLa cells.

Antibodies raised against the C-terminus and N-terminus region of gamma gamma enolase, as well as a polyclonal antibody raised against bovine brain gamma gamma enolase, were used to study the distribution of this glycolytic enzyme during the cell cycle in HeLa cells. Enolase was found to be present throughout the cytoplasm of both interphase and dividing cells. In addition, a portion of cellular enolase was detected at the centrosome throughout the cell cycle. The capacity of glycolytic enzymes to play a structural as well as a glycolytic role suggests that the presence of enolase at the centrosome may be correlated with the organization of both the interphase cytoskeleton and the mitotic spindle.     

Hormone-free mouse glucocorticoid receptors overexpressed in Chinese hamster ovary cells are localized to the nucleus and are associated with both hsp70 and hsp90

In this work, we examine the cellular localization and protein interactions of mouse glucocorticoid receptors that have been overexpressed in Chinese hamster ovary (CHO) cells (Hirst, M. A., Northrop, J. P., Danielsen, M., and Ringold, G. M. (1990) Mol. Endocrinol. 4, 162-170). We demonstrate that wild-type unliganded mouse glucocorticoid receptor, which is expressed in CHO cells to a level approximately 10 times that of L cells, is localized entirely to the nucleus by indirect immunofluorescence with the BuGR antireceptor monoclonal antibody. Overexpressed receptors that have either no hormone binding activity or no DNA binding activity because of point mutations also localize to the nucleus, providing genetic proof that the nuclear localization cannot reflect a steroid-mediated shift of the receptor from the cytoplasm to the nucleus and that DNA binding activity is not required for nuclear localization. Like unliganded progesterone receptors, which also associate in a loosely bound "docking" complex with the nucleus, the mouse glucocorticoid receptor overexpressed in CHO cells is associated with both hsp90 and hsp70. This is in contrast to the untransformed mouse glucocorticoid receptor in L cell cytosol, which is associated with hsp90 but not hsp70. The difference in hsp70 association between cell types could reflect overexpression of the receptor in CHO cells. However, like receptors in CHO cells selected for very high levels of overexpression, receptors in CHO cells selected for an intermediate level of receptor expression that is comparable to that of L cells are also bound to hsp70. This observation argues against an explanation of hsp70 association based purely on receptor overexpression, and we speculate that association of the unliganded glucocorticoid receptor with hsp70 might be a consequence of its nuclear localization in the CHO cells. Although there are differences between the mouse receptor in CHO cells and L cells, the nuclear localization signal of the untransformed mouse receptor reacts equivalently with the AP64 antibody against NL1 in cytosols prepared from both cell types.     

Myosin II activity is not essential for recruitment of myosin II to the furrow in dividing HeLa cells

To elucidate whether phosphorylation of myosin II regulatory light chain (MRLC) is essential for myosin II recruitment to the furrow during cytokinesis, HeLa cells transfected with three types of GFP-tagged recombinant MRLCs, wild-type MRLC, non-phosphorylated form of MRLC, and phosphorylated form of MRLC, were examined. Living cell-imaging showed that both phosphorylated and non-phosphorylated form of MRLCs were recruited to the equator at the same time after anaphase onset, suggesting that phosphorylation of MRLC is not responsible for recruitment of myosin II to the equator. Moreover, the treatment with an inhibitor of myosin II activity, blebbistatin, induced no effect on recruitment of those three recombinant MRLCs. During cytokinesis, phosphorylated but not non-phosphorylated form of MRLC was retained in the equator. These results suggest that phosphorylation of MRLC is essential for retainment of myosin II in the furrow but not for initial recruitment of myosin II to the furrow in dividing HeLa cells.     

Apoprotein B degradation is promoted by the molecular chaperones hsp90 and hsp70

Apoprotein B (apoB) is the major protein of liver-derived atherogenic lipoproteins. The net production of apoB can be regulated by presecretory degradation mediated by the ubiquitin-proteasome pathway and cytosolic hsp70. To further explore the mechanisms of apoB degradation, we have established a cell-free system in which degradation can be faithfully recapitulated. Human apoB48 synthesized in vitro was translocated into microsomes, glycosylated, and ubiquitinylated. Subsequent incubation with rat hepatic cytosol led to proteasome-mediated degradation. To explore whether hsp90 is required for apoB degradation, geldanamycin (GA) was added during the degradation assay. GA increased the recovery of microsomal apoB48 approximately 3-fold and disrupted the interaction between hsp90 and apoB48. Confirming the hsp90 effect in the cell-free system, we also found that transfection of hsp90 cDNA into rat hepatoma cells enhanced apoB48 degradation. Finally, apoB48 degradation was reconstituted in vitro using cytosol prepared from wild type yeast. Notably, degradation was attenuated when apoB48-containing microsomes were incubated with cytosol supplemented with GA or with cytosol prepared from yeast strains with mutations in the homologues of mammalian hsp70 and hsp90. Overall, our data suggest that hsp90 facilitates the interaction between endoplasmic reticulum-associated apoB and components of the proteasomal pathway, perhaps in cooperation with hsp70.     

Synthesis of trypanosome hsp70 mRNA is resistant to disruption of trans-splicing by heat shock

Synthesis of mRNA in trypanosomes involves an apparent trans-splicing reaction whereby a common 39-nucleotide mini-exon sequence is joined to the protein-coding exon of a mRNA precursor. We have previously shown (Muhich, M. L., and Boothroyd, J. C. (1988) Mol. Cell. Biol. 8, 3837-3846) that the trans-splicing pathway of Trypanosoma brucei is sensitive to disruption by severe heat shock. Here we demonstrate that the synthesis of heat shock protein 70 (hsp 70) mRNA in T. brucei is apparently resistant to the heat-induced disruption of splicing. The 5'-ends of hsp 70 mRNAs are shown to be identical for molecules synthesized at either normal or heat shock temperatures, and in both cases, the 5'-terminal mini-exon sequence is present. These results suggest that T. brucei has evolved a mechanism which directly compensates for the deleterious effects of heat shock on trans-splicing, one which allows for the continued mini-exon-dependent trans-splicing of selected pre-mRNAs.     

CP60: a microtubule-associated protein that is localized to the centrosome in a cell cycle-specific manner.

DMAP190 is a microtubule-associated protein from Drosophila that is localized to the centrosome. In a previous study, we used affinity chromatography to identify proteins that interact with DMAP190, and identified a 60-kDa protein that we named DMAP60 (Kellogg and Alberts, 1992). Like DMAP190, DMAP60 interacts with microtubules and is localized to the centrosome, and the two proteins associate as part of a multiprotein complex. We now report the cloning and sequencing of the cDNA encoding DMAP60. The amino acid sequence of DMAP60 is not homologous to any protein in the database, although it contains six consensus sites for phosphorylation by cyclin-dependent kinases. As judged by in situ hybridization, the gene for DMAP60 maps to chromosomal region 46A. In agreement with others working on Drosophila centrosomal proteins, we have changed the names for DMAP190 and DMAP60 to CP190 and CP60, respectively, to give these proteins a consistent nomenclature. Antibodies that recognize CP60 reveal that it is localized to the centrosome in a cell cycle-dependent manner. The amount of CP60 at the centrosome is maximal during anaphase and telophase, and then drops dramatically during late telophase or early interphase. This dramatic disappearance of CP60 may be due to specific proteolysis, because CP60 contains a sequence of amino acids similar to the "destruction box" that targets cyclins for proteolysis at the end of mitosis. Starting with nuclear cycle 12, CP60 and CP190 are both found in the nucleus during interphase. CP60 isolated from Drosophila embryos is highly phosphorylated, and dephosphorylated CP60 is a good substrate for cyclin B/p34cdc2 kinase complexes. A second kinase activity capable of phosphorylating CP60 is present in the CP60/CP190 multiprotein complex. We find that bacterially expressed CP60 binds to purified microtubules, and this binding is blocked by CP60 phosphorylation.     

The hsp90 cochaperone p23 is the limiting component of the multiprotein hsp90/hsp70-based chaperone system in vivo where it acts to stabilize the client protein: hsp90 complex

A variety of signaling proteins form heterocomplexes with and are regulated by the heat shock protein chaperone hsp90. These complexes are formed by a multiprotein machinery, including hsp90 and hsp70 as essential and abundant components and Hop, hsp40, and p23 as non-essential cochaperones that are present in much lower abundance in cells. Overexpression of signaling proteins can overwhelm the capacity of this machinery to properly assemble heterocomplexes with hsp90. Here, we show that the limiting component of this assembly machinery in vitro in reticulocyte lysate and in vivo in Sf9 cells is p23. Only a fraction of glucocorticoid receptors (GR) overexpressed in Sf9 cells are in heterocomplex with hsp90 and have steroid binding activity, with the majority of the receptors present as both insoluble and cytosolic GR aggregates. Coexpression of p23 with the GR increases the proportion of cytosolic receptors that are in stable GR.hsp90 heterocomplexes with steroid binding activity, a strictly hsp90-dependent activity for the GR. Coexpression of p23 eliminates the insoluble GR aggregates and shifts the cytosolic receptor from very large aggregates without steroid binding activity to approximately 600-kDa heterocomplexes with steroid binding activity. These data lead us to conclude that p23 acts in vivo to stabilize hsp90 binding to client protein.     

Hsp70 protects mitotic cells against heat-induced centrosome damage and division abnormalities

The effect of heat shock on centrosomes has been mainly studied in interphase cells. Centrosomes play a key role in proper segregation of DNA during mitosis. However, the direct effect and consequences of heat shock on mitotic cells and a possible cellular defense system against proteotoxic stress during mitosis have not been described in detail. Here, we show that mild heat shock, applied during mitosis, causes loss of dynamitin/p50 antibody staining from centrosomes and kinetochores. In addition, it induces division errors in most cells and in the remaining cells progression through mitosis is delayed. Expression of heat shock protein (Hsp)70 protects against most heat-induced division abnormalities. On heat shock, Hsp70 is rapidly recruited to mitotic centrosomes and normal progression through mitosis is observed immediately after release of Hsp70 from centrosomes. In addition, Hsp70 expression coincides with restoration of dynamitin/p50 antibody staining at centrosomes but not at kinetochores. Our data show that during mitosis, centrosomes are particularly affected resulting in abnormal mitosis. Hsp70 is sufficient to protect against most division abnormalities, demonstrating the involvement of Hsp70 in a repair mechanism of heat-damaged mitotic centrosomes.     

The serine 2481-autophosphorylated form of mammalian Target Of Rapamycin (mTOR) is localized to midzone and midbody in dividing cancer cells

Using a high-resolution, automated confocal high-content imaging system, we investigated the sub-cellular localization of the Serine 2481-autophosphorylated form of mTOR (PP-mTOR^Ser2481) during mitosis and cytokinesis in human cancer cells. PP-mTOR^Ser2481 exhibited a punctate nuclear distribution in interphase cancer cells, with the number of PP-mTOR^Ser2481 nuclear speckles positively relating with the proliferative capacity of cancer cells. PP-mTOR^Ser2481 expression dynamically rearranged within the cytoplasm in a close association near and between separating chromosomes during early stages of mitosis. Towards the end of anaphase and in telophase, PP-mTOR^Ser2481 drastically focused on the midzone and ultimately in the centre of the midbody at the presumptive cleavage furrow. In cells at cytokinesis, PP-mTOR^Ser2481 appeared as a doublet facing each other at the apical ends of two daughter cells. Three-dimensional analysis confirmed that PP-mTOR^Ser2481 positioned at a ring structure wrapped round by microtubule bundles to connect daughter cells. These results reveal for the first time that PP-mTOR^Ser2481 may be unexpectedly involved in the terminal stages of cytokinesis.     

Decreased heat- and tumor necrosis factor-mediated hsp28 phosphorylation in thermotolerant HeLa cells

Heat shock or tumor necrosis factor rapidly stimulated the phosphorylation of the mammalian low molecular weight stress protein hsp28. We have found that both phenomena are greatly decreased in cells which are made tolerant to heat. This observation correlated with a better survival of thermotolerant cells exposed to either heat or TNF treatment. The results suggest that the phosphorylation of hsp28 may be linked to the resistance of the cells to the deleterious effects induced by either heat or a mediator of inflammation such as TNF.     

Starvation promotes nuclear accumulation of the hsp70 Ssa4p in yeast cells

Nuclear import of proteins that are too large to passively enter the nucleus requires soluble factors, energy, and a nuclear localization signal (NLS). Nuclear protein transport can be regulated, and different forms of stress affect nucleocytoplasmic trafficking. As such, import of proteins containing a classical NLS is inhibited in starving yeast cells. In contrast, the hsp70 Ssa4p concentrates in nuclei upon starvation. Nuclear concentration of Ssa4p in starving cells is reversible, and transfer of stationary phase cells to fresh medium induces Ssa4p nuclear export. This export reaction represents an active process that is sensitive to oxidative stress. In starving cells, the N-terminal domain of Ssa4p mediates Ssa4p nuclear accumulation, and a short hydrophobic sequence, termed Star (for starvation), is sufficient to localize the reporter proteins green fluorescent protein or beta-galactosidase to nuclei. To determine whether nuclear accumulation of Star-beta-galactosidase depends on a specific nuclear carrier, we have analyzed its distribution in mutant yeast strains that carry a deletion of a single beta-importin gene. With this assay we have identified Nmd5p as a beta-importin required to concentrate Star-beta-galactosidase in nuclei when cells enter stationary phase.     

Characterisation of a pea hsp70 gene which is both developmentally and stress-regulated

A pea pod cDNA library was screened for sequences specific to lignifying tissue. A cDNA clone (pLP19) encoding the C-terminal region of a hsp70 heat shock protein hybridised only to pod mRNA from pea lines where pod lignification occurred. Expression of pLP19 was induced by heat shock in leaves, stems and roots of pea and chickpea plants. Four different poly(A) addition sites were observed in cDNAs derived from the same gene as pLP19. This gene was fully sequenced; unlike most hsp70 genes, it contains no introns. The 5-flanking sequence contains heat shock elements and other potential regulatory sequences.     

Alsin is partially associated with centrosome in human cells

Mutations in the ALS2 gene has recently been linked to cases of juvenile amyotrophic lateral sclerosis, juvenile primary lateral sclerosis and ascending hereditary spastic paralysis. All reported mutations predict the production of truncated forms of Alsin suggesting a loss of function mechanism for these motor neuron disorders. Here we used the tetracycline-regulated expression system to overexpress the full-length and truncated forms of Alsin in different cell lines. Alsin overexpression caused severe phenotypic changes in monkey COS-7 cells including the enlargement and accumulation of early endosomes, impairment of mitochondria trafficking and fragmentation of the Golgi apparatus. Our results further demonstrate the requirement of the Alsin VPS9 domain for occurrence of the vacuolation process and the role of Alsin as a guanine nucleotide exchange factor for Rab5. Transfected human SW13 cells exhibited an unexpected centrosomal localization for Alsin that was linked to the presence of the c-terminal part of the protein. Immunofluorescence staining revealed a colocalization of Alsin with the centrosomal markers gamma-tubulin and A kinase anchoring protein (AKAP-450). Similar results were obtained with human LA-N-2 and SK-N-SH neuronal cells. Moreover endogenous Alsin was detected in a centrosome preparation purified from human cortical brain. Considering the crucial role of centrosome in the production of microtubules required for intracellular transport, these findings are of potential relevance for unravelling the disease mechanisms linked to Alsin mutations.     

The chaperone function of hsp70 is required for protection against stress-induced apoptosis

Cellular stress can trigger a process of self-destruction known as apoptosis. Cells can also respond to stress by adaptive changes that increase their ability to tolerate normally lethal conditions. Expression of the major heat-inducible protein hsp70 protects cells from heat-induced apoptosis. hsp70 has been reported to act in some situations upstream or downstream of caspase activation, and its protective effects have been said to be either dependent on or independent of its ability to inhibit JNK activation. Purified hsp70 has been shown to block procaspase processing in vitro but is unable to inhibit the activity of active caspase 3. Since some aspects of hsp70 function can occur in the absence of its chaperone activity, we examined whether hsp70 lacking its ATPase domain or the C-terminal EEVD sequence that is essential for peptide binding was required for the prevention of apoptosis. We generated stable cell lines with tetracycline-regulated expression of hsp70, hsc70, and chaperone-defective hsp70 mutants lacking the ATPase domain or the C-terminal EEVD sequence or containing AAAA in place of EEVD. Overexpression of hsp70 or hsc70 protected cells from heat shock-induced cell death by preventing the processing of procaspases 9 and 3. This required the chaperone function of hsp70 since hsp70 mutant proteins did not prevent procaspase processing or provide protection from apoptosis. JNK activation was inhibited by both hsp70 and hsc70 and by each of the hsp70 domain mutant proteins. The chaperoning activity of hsp70 is therefore not required for inhibition of JNK activation, and JNK inhibition was not sufficient for the prevention of apoptosis. Release of cytochrome c from mitochondria was inhibited in cells expressing full-length hsp70 but not in cells expressing the protein with ATPase deleted. Together with the recently identified ability of hsp70 to inhibit cytochrome c-mediated procaspase 9 processing in vitro, these data demonstrate that hsp70 can affect the apoptotic pathway at the levels of both cytochrome c release and initiator caspase activation and that the chaperone function of hsp70 is required for these effects.