A role for the cytoskeleton in heat-shock-mediated thermoprotection of locust neuromuscular junctions

A prior hyperthermic stress (heat shock) can induce thermoprotection of neuromuscular transmission in Locusta migratoria extensor tibiae muscle measured 4 h after the onset of the heat shock. It is not clear what effect an acute hyperthermic stress may have on the nervous system's ability to tolerate thermal stress, that is, before increased expression of heat-shock proteins. We found that over consecutive thermal stress tests, failure temperature was not altered in either heat-shock or control animals. This suggests that protective mechanisms are not established in the short term (within one hour). Various members of the heat-shock protein family interact with elements of the cytoskeleton. We found that preexposure of the preparation to cytoskeletal stabilizing drugs induced thermoprotection, while preexposure to cytoskeletal disrupting drugs disrupted the ability to confer and maintain thermoprotection. We conclude that thermoprotection relies on a stable cytoskeleton and suggest that members of the heat shock protein family are involved.     

Synaptic thermoprotection in a desert‐dwelling Drosophila species

Synaptic transmission is a critical mechanism for transferring information from the nervous system to the body. Environmental stress, such as extreme temperature, can disrupt synaptic transmission and result in death. Previous work on larval Drosophila has shown that prior heat‐shock exposure protects synaptic transmission against failure during subsequent thermal stress. This induced thermoprotection has been ascribed to an up‐regulation of the inducible heat‐shock protein, Hsp70. However, the mechanisms mediating natural thermoprotection in the wild are unknown. We compared synaptic thermosensitivity between D. melanogaster and a desert species, D. arizonae. Synaptic thermosensitivity and the functional limits of the related locomotor behavior differed significantly between closely related, albeit ecologically distinct species. Locomotory behavior of wandering third instar D. arizonae larvae was less thermosensitive and the upper temperature limit of locomotory function exceeded that of D. melanogaster by 6°C. Behavioral results corresponded with significantly lower synaptic thermosensitivity at the neuromuscular junction in D. arizonae. Prior heat‐shock protected only D. melanogaster by increasing relative excitatory junctional potential (EJP) duration, the time required for EJP failure at 40°C, and the incidence of EJP recovery following heat‐induced failure. Hsp70 induction profiles following heat‐shock demonstrate up‐regulation of inducible Hsp70 in D. melanogaster but not in D. arizonae. However, expression of Hsp70 under control conditions is greater in D. arizonae. These results suggest that the mechanisms of natural thermoprotection involve an increase in baseline Hsp70 expression. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Role for calcium in heat shock‐mediated synaptic thermoprotection in Drosophila larvae

Chemical synaptic transmission is the mechanism for fast, excitation‐coupled information transfer between neurons. Previous work in larval Drosophila has shown that transmission at synaptic boutons is protected by heat shock exposure from subsequent thermal stress through pre‐ and postsynaptic modifications. This protective effect has been, at least partially, ascribed to an up‐regulation in the inducible heat shock protein, hsp70. Effects of hsp70 are correlated with changes to intracellular calcium handling, and the dynamics of intracellular calcium regulate synaptic transmission. Consistent with such a relationship, synaptic plasticity increases at locust neuromuscular junctions following heat shock, suggesting an effect of heat shock on residual presynaptic calcium. Intracellular recording from single abdominal muscle fibers of Drosophila larvae showed that prior heat shock imparts thermoprotection by increasing the upper temperature limit for synaptic transmission. Heat shock exposure enhances short‐term synaptic plasticity and increases its thermosensitivity. Increasing extracellular calcium levels eliminates the physiological differences between control and heat shock preparations; excess calcium itself induces thermoprotection at elevated concentrations. These data support the hypothesis that stress‐induced neuroprotection at the nerve terminal acts, at least partially, through an alteration to the physiological effects of residual presynaptic calcium. © 2003 Wiley Periodicals, Inc. J Neurobiol 56: 360–371, 2003     

Cytoskeletal stability and heat shock-mediated thermoprotection of central pattern generation in Locusta migratoria

Prior exposure to extreme temperatures can induce thermoprotection in migratory locusts, which is important for survival in their natural environment. An important motor activity that needs to be protected is ventilation. The mechanism underlying heat shock is not fully understood, and our goal was to test the idea that cytoskeletal stability is critical for such thermoprotection. Cytoskeletal stabilizers (concanavalin A) and destabilizers (colchicine) were bath-applied in semi-intact locust preparations in both control (C) and pre-treated heat-shocked (3 h, 45 degrees C) animals. We measured parameters of the ventilatory motor pattern during maintained high temperature (43 degrees C) and recorded the times taken for motor pattern generation to fail and then recover on returning to room temperature. We found that concanavalin A mimicked the effects of a prior heat stress in control animals by increasing time to failure and decreasing time to recovery of motor pattern generation. However, colchicine destroyed protection in heat-shocked animals by decreasing time to failure and increasing time to recovery. Our findings confirm that the cytoskeleton has a mechanistic role in preserving neural function at high temperatures, possibly through stabilizing ion channels and other integral membrane proteins (e.g. Na(+)/K(+) ATPase) and their interactions with heat shock proteins.     

Specific protein homeostatic functions of small heat‐shock proteins increase lifespan

During aging, oxidized, misfolded, and aggregated proteins accumulate in cells, while the capacity to deal with protein damage declines severely. To cope with the toxicity of damaged proteins, cells rely on protein quality control networks, in particular proteins belonging to the family of heat‐shock proteins (HSPs). As safeguards of the cellular proteome, HSPs assist in protein folding and prevent accumulation of damaged, misfolded proteins. Here, we compared the capacity of all Drosophila melanogaster small HSP family members for their ability to assist in refolding stress‐denatured substrates and/or to prevent aggregation of disease‐associated misfolded proteins. We identified CG14207 as a novel and potent small HSP member that exclusively assisted in HSP70‐dependent refolding of stress‐denatured proteins. Furthermore, we report that HSP67BC, which has no role in protein refolding, was the most effective small HSP preventing toxic protein aggregation in an HSP70‐independent manner. Importantly, overexpression of both CG14207 and HSP67BC in Drosophila leads to a mild increase in lifespan, demonstrating that increased levels of functionally diverse small HSPs can promote longevity in vivo.     

Heat shock gene expression and cytoskeletal alterations in mouse neuroblastoma cells

The cytoskeleton of neuroblastoma cells, clone Neuro 2A, is altered by two stress conditions: heat shock and arsenite treatment. Microtubules are reorganized, intermediate filaments are aggregated around the nucleus, and the number of stress fibers is reduced. Since both stress modalities induce similar cytoskeletal alterations, no thermic denaturation of one or more cytoskeletal components can be involved in this process. Heat shock proteins are induced both by heat and by arsenite. However, cells treated with arsenite synthesize hsp28 which is not detected in heat-treated cells. Synthesis of all hsps is prevented by addition of actinomycin D or cycloheximide. Under these conditions no alterations are observed in the organization of microtubules and intermediate filaments during heat or arsenite treatment. However, these drugs are not able to prevent the rapid loss of stress fibers. A re-formation of the cytoskeleton during the recovery period proceeds within 3 h and is also found to occur in the presence of a protein synthesis inhibitor. These data suggest that reorganization of microtubules and intermediate filaments during a stress treatment requires the synthesis of a new protein(s), probably hsp(s).     

Cell-type-specific disruption and recovery of the cytoskeleton in Arabidopsis thaliana epidermal root cells upon heat shock stress

Summary. The cytoskeleton in plant cells plays an important role in controlling cell shape and mediating intracellular signalling. However, almost nothing is known about the reactions of cytoskeletal elements to heat stress, which represents one of the major environmental challenges for plants. Here we show that living epidermal root cells of Arabidopsis thaliana could cope with short-term heat shock stress showing disruption and subsequent recovery of microtubules and actin microfilaments in a time-dependent manner. Time-lapse imaging revealed a very dynamic behavior of both cytoskeletal elements including transient depolymerization and disassembly upon heat shock (40–41 °C) followed by full recovery at room temperature (20 °C) within 1–3 h. Reaction of microtubules, but not actin filaments, to heat shock was dependent on cell type and developmental stage. On the other hand, recovery of actin filaments, but not microtubules, from heat shock stress was dependent on the same parameters. The relevance of this adaptive cytoskeletal behavior to intracellular signalling is discussed. Correspondence and reprints: Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, 53115 Bonn, Federal Republic of Germany.     

Role for calcium in heat shock-mediated synaptic thermoprotection in Drosophila larvae

Chemical synaptic transmission is the mechanism for fast, excitation-coupled information transfer between neurons. Previous work in larval Drosophila has shown that transmission at synaptic boutons is protected by heat shock exposure from subsequent thermal stress through pre- and postsynaptic modifications. This protective effect has been, at least partially, ascribed to an up-regulation in the inducible heat shock protein, hsp70. Effects of hsp70 are correlated with changes to intracellular calcium handling, and the dynamics of intracellular calcium regulate synaptic transmission. Consistent with such a relationship, synaptic plasticity increases at locust neuromuscular junctions following heat shock, suggesting an effect of heat shock on residual presynaptic calcium. Intracellular recording from single abdominal muscle fibers of Drosophila larvae showed that prior heat shock imparts thermoprotection by increasing the upper temperature limit for synaptic transmission. Heat shock exposure enhances short-term synaptic plasticity and increases its thermosensitivity. Increasing extracellular calcium levels eliminates the physiological differences between control and heat shock preparations; excess calcium itself induces thermoprotection at elevated concentrations. These data support the hypothesis that stress-induced neuroprotection at the nerve terminal acts, at least partially, through an alteration to the physiological effects of residual presynaptic calcium.     

Hyperthermic stress stimulates the association of both constitutive and inducible isoforms of 70 kDa heat shock protein with rat liver glucocorticoid receptor

Glucocorticoid hormone receptor exists in the cytoplasm of target cells in the form of dynamic multiprotein heterocomplexes with heat shock proteins Hsp90 and Hsp70, and additional components of the molecular chaperone machinery. Whole body hyperthermic stress was previously shown to induce alterations in protein composition of these complexes increasing the share of Hsp70, but participation of individual Hsp70 family members was not investigated. In the present study the association of glucocorticoid receptor with constitutive and inducible forms of Hsp70 in the liver cytosol of rats exposed to 41 degrees C whole body hyperthermic stress was examined. Immunoprecipitation of glucocorticoid receptor heterocomplexes by monoclonal anti-receptor antibody (BuGR2) followed by quantitative immunoblotting revealed the presence of both nucleocytoplasmic Hsp70 family members, constitutive--Hsc70 and inducible--Hsp72, within the complexes. Immediately after the stress only Hsc70 was found in association with glucocorticoid receptor. However, after the induction of Hsp72 by stress, its appearance within the glucocorticoid receptor heterocomplexes was also recorded and the presence of both Hsp70 forms within the heterocomplexes was evident by the end of examined 24h period after the stress. This study confirms that heat stress affects protein composition of rat liver glucocorticoid receptor heterocomplexes increasing the share of Hsp70 and shows that this increase could be equally ascribed to constitutive and inducible forms of Hsp70.     

Heat shock proteins do not provide thermoprotection to normal cellular protein synthesis, α-amylase mRNA and endoplasmic reticulum lamellae in barley aleurone layers

Heat shock in barley ( Hordeum vulgare L. cv. Himalaya) aleurone layers induces the synthesis of heat shock proteins (hsps) and suppresses the synthesis and secretion of α-amylase, the principal secretory protein. This is accompanied by the destabilization of α-amylase mRNA and a concomitant dissociation of ER lamellae. In the absence of heat shock α-amylase mRNA is extremely stable (Belanger et al. 1986. Proc. Natl. Acad. Sci. USA 83: 1354–1358). In most organisms there is a direct correlation between the synthesis of hsps and thermotolerance. The ability of hsps to provide thermoprotection to secretory protein synthesis, α-amylase mRNA and ER lamellae was analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of pulse-chased, [³⁵S]-methionine-labeled proteins revealed that the half-life of hsps in barley aleurone cells recovering from heat shock was approximately 12 h. Within approximately 6 h, there was a recovery of α-amylase mRNA and a reformation of ER lamellae. Heat shock protein synthesis was induced by either heat shock (40°C) or arsenite, the cells were allowed to recover for 8 h, then were re-exposed to heat shock. Results from SDS-PAGE showed that, despite the presence of hsps, α-amylase synthesis was suppressed. Northern blot hybridizations showed that α-amylase mRNA levels were reduced in heat-shocked tissues. Transmission electron microscopy demonstrated that ER lamellar structures were dissociated. The synthesis of hsps did not enable barley aleurone cells to sustain the synthesis of any proteins at lethal temperature. In contrast, similar conditions established thermotolerance and provided thermoprotection to protein synthesis in germinating barley embryos. Our findings suggest that the aleurone layer does not become thermotolerant following the induction of hsp synthesis.     

Secretion of the heat shock proteins HSP70 and HSC70 by baby hamster kidney (BHK‐21) cells

The HSPs (heat‐shock proteins) of the 70‐kDa family, the constitutively expressed HSC70 (cognate 70‐kDa heat‐shock protein) and the stress‐inducible HSP70 (stress‐inducible 70‐kDa heat‐shock protein), have been reported to be actively secreted by various cell types. The mechanisms of the release of these HSPs are obscure, since they possess no consensus secretory signal sequence. We showed that baby hamster kidney (BHK‐21) cells released HSP70 and HSC70 in a serum‐free medium and that this process was the result of an active secretion of HSPs rather than the non‐specific release of the proteins due to cell death. It was found that the secretion of HSP70 and HSC70 is independent of de novo protein synthesis. BFA (Brefeldin A) did not inhibit the basal secretion of HSPs, indicating that the secretion of HSP70 and HSC70 from cells occurs by a non‐classical pathway. Exosomes did not contribute to the secretion of HSP70 and HSC70 by cells. MBC (methyl‐β‐cyclodextrin), a substance that disrupts the lipid raft organization, considerably reduced the secretion of both HSPs, indicating that lipid rafts are involved in the secretion of HSP70 and HSC70 by BHK‐21 cells. The results suggest that HSP70 and HSC70 are actively secreted by BHK‐21 cells in a serum‐free medium through a non‐classical pathway in which lipid rafts play an important role.     

Differential proteomic response to heat stress in thermal Agrostis scabra and heat‐sensitive Agrostis stolonifera

Knowledge of heat‐responsive proteins is critical for further understanding of the molecular mechanisms of heat tolerance. The objective of this study was to compare proteins differentially expressed in two C₃ grass species contrasting in heat tolerance, heat‐tolerant thermal Agrostis scabra and heat‐sensitive Agrostis stolonifera L., and to identify heat‐responsive proteins for short‐ and long‐term responses. Plants were exposed to 20/15°C (day/night, control) or 40/35°C (day/night, heat stress) in growth chambers. Leaves were harvested at 2 and 10 days after temperature treatment. Proteins were extracted and separated by fluorescence difference gel electrophoresis (DIGE). Thermal A. scabra had superior heat tolerance than A. stolonifera, as indicated by the maintenance of higher chlorophyll content and photochemical efficiency under heat stress. The two‐dimensional difference electrophoresis detected 68 heat‐responsive proteins in the two species. Thermal A. scabra had more protein spots either down‐ or up‐regulated at 2 days of heat stress, but fewer protein spots were altered at 10 days of heat stress compared with A. stolonifera. Many protein spots exhibited transient down‐regulation in thermal A. scabra (only at 2 days of heat treatment), whereas down‐regulation of many proteins was also found at 10 days of heat treatment in A. stolonifera, which suggested that protein metabolism in thermal A. scabra might acclimate to heat stress more rapidly than those in A. stolonifera. The sequences of 56 differentially expressed protein spots were identified using mass spectrometry. The results suggest that the maintenance or less severe down‐regulation of proteins during long‐term (10 days) heat stress may contribute to the superior heat tolerance in thermal A. scabra, including those involved in photosynthesis [RuBisCo, RuBisCo activase, chloroplastic glyceraldehydes‐3‐phosphate dehydrogenase (GAPDH), chloroplastic aldolase, oxygen‐evolving complex, photosystem I subunits], dark respiration (cytosolic GAPDH, cytoplasmic aldolase, malate dehydrogenase, hydroxypyruvate reductase, sedoheptulose‐1,7‐bisphosphatase), photorespiration [(hydroxypyruvate reductase, alanine aminotransferase (AlaAT), hydroxymethyltransferase (SHMT), glycine decarboxylase (GDC)], as well as heat and oxidative stress protection [heat shock cognate (HSC) 70 and FtsH‐like protein].