Small heat shock proteins of Drosophila associate with the cytoskeleton.

Abstract

Fractionation of heat-shocked Drosophila melanogaster Kc cells reveals that both the small heat shock proteins (hsp28, -26, -23, and -22) and vnmentin-like intermediate filament proteins (EFPs) are abundantly represented in the nuclear fraction. Cofractionation of the IFPs with nuclei is due to the collapse of the IFP network against the nucleus upon heat shock, raising the possibility that cofractionation of the small hsps is by a similar mehanis. Indirect Immunofluorescence supports this possibility. In salivary glands, both the hsps and the IFPs are cytoplasmic after mild-to-moderate heat shocks and only enter the nucleus upon severe-indeed, lethal-shocks. Double-label experiments with Schneider line 2 cells show that the IFPs and small hsps colocalize to the some perinuclear aggregates in 70% of the cells examined. Thus, the small hsps are associated with the cytoskeleton rather than with nuclear structures.The heat shock response is a ubiquitous cellular response to physiological stress characterized by the rapid induction of a small number of heat shock proteins (hsps; reviewed in refs. 1-3). The extreme conservation of the response and of the hsps themselves suggests that these proteins carry out essential functions. However, aside from their role in the development of thermotolerance-the ability to withstand a second heat shock at an otherwise lethal temperature (4-10)-little is known about their function(s). Thus far, the most progress has been made for hsp70. In yeast, replacement of several members of the hsp7O gene family with mutant genes indicates a role for hsp70 in growth at high temperatures (11). Likewise, mutants in dnaK (the Escherichia coli counterpart ofhsp70) are temperature-sensitive for growth (12) and are unable to recover normal protein synthesis following heat shock (13). hsp70 appears to play a similar role in Drosophila (14, 15) and apparently carries out this function within the nucleus (16).As a means of addressing the function of the low molecular weight family of hsps, several investigators have examined the intracellular distribution of these proteins. Several reports have argued for an association with chromatin and nucleoli (8,17). Yet, others appear to rule out association with chromatin and suggest that these proteins are part of the nuclear matrix (18,19 (20)(21)(22)(23). This cofractionation appears to be due to the collapse of the IFP network against the nucleus within 5 min of heat shock (20-23). In order to assess the contribution of the collapse of the IF cytoskeleton on the localization of the small hsps, we have reinvestigated the intracellular distribution of the small hsps ofDrosophila by biochemical fractionation and indirect immunofluorescence. The data best support association of these proteins with the IF cytoskeleton. For heat shock, cells were incubated at 370C for 15 min prior to harvest. Cells were rinsed with Echalier's medium, concentrated to 1 ml, and labeled with 50 ,uCi (1 Ci = 37 GBq) of [35S]methionine (Amersham) at 250C or 37TC for 1 hr. T...

Dynamic changes in the structure and intracellular locale of the mammalian low-molecular-weight heat shock protein.

“…It has been suggested that the distribution of the low-MW HSPs in the detergent-insoluble nuclear-cytoskeletal fraction after heat shock is a consequence of such filament collapse (30). Although we cannot rule out this possibility entirely, we have shown here that treatment of cells with various other agents that promote the collapse of the intermediate filaments does not result in the redistribution of the 28-kDa protein into the insoluble fraction.…”

Cytoplasmic heat shock granules are formed from precursor particles and are associated with a specific set of mRNAs.

“…In contrast to earlier reports on the nuclear localization of small HSPs in heat-shocked cells of D. melanogaster (5,35,58,62) and plants (36) which were based on cell fractionation procedures, it is now firmly established that the mass of small HSPs in D. melanogaster (2,19,34), vertebrates (8,17), and plants (47) is in fact contained in cytoplasmic aggregates concentrated in the perinuclear region. A clue to the reinvestigation of previous data was the detection and biochemical characterization of these aggregates (heat shock granules [HSGs]) in tomato cell cultures (42,43,46,47).…”

“…1) reveal three classes: (i) the bulk of mRNAs, which are nontranslated during heat shock but are preserved (Fig. 1H); (ii) a few control mRNAs whose translation continues during heat shock, e.g., mRNAs cod- (18,57 (14,17,22,34,(64)(65)(66) (17,34). The close location is impressively illusosition of prosomes (1) and pre-HSGs (2) trated by a freeze fracture image of chicken cells showing .…”

Cytoplasmic Distribution of Heat Shock Proteins in Soybean

“…Nover et al (20) postulated that hs granules are unique to plants as no structure analogous to hs granules has been detected in animal systems. Unlike the cell-wide distribution of the low mol wt hs proteins in plants (19), the low mol wt hs proteins of Drosophila are associated with elements of the cytoskeleton during hs (13). During recovery, they are found in 20S RNP particles similar to prosomes, a class of scRNP particles implicated in the negative control of mRNA translation (3).…”

“…6). Sedimenting in advance of the polysome peak was a broad shoulder of absorptive material presumably representing the hs granules (fractions [6][7][8][9][10][11][12][13][14]. This material contained the majority of the radioactivity.…”