The basal and more or less universal regulation of the HS response operates through the transcriptional induction of HS genes by the activated HS factor (Morimoto et al 1994
; Wu et al 1994
). However, it is becoming increasingly clear that there must exist additional regulatory mechanisms that cause some cells to respond to stress in a subtle or more noticeably different manner (Feder and Hoffmann 1999
; Lakhotia 2001a
; Lakhotia and Prasanth 2002
). The HS response in MTs of Drosophila
larvae is very unusual because, as reported earlier (Lakhotia and Singh 1989
), none of the typical Hsps are induced after HS in MT, but a different set of polypeptides is induced. Among these, a 64-kDa polypeptide belonging to the Hsp60 family was found to be maximally induced (Lakhotia and Singh 1989
). Unlike the other tissues, synthesis of Hsp70, and presumably of the other common Hsps, in larval MTs is induced only later during recovery (Krebs and Feder 1997
Present results reveal 2 significant aspects of the HS response in MT cells of Drosophila larvae. The first relates to the unchanging levels of Hsp60 in the MT cells, whether the HS-induced new synthesis is allowed or prevented by transcriptional or translational inhibitors. The second relates to immediate activation of the hsp70 genes after HS but a delayed appearance of Hsp70.
As initially reported by Lakhotia and Singh (1989)
and reconfirmed in the present study, 35
S-methionine incorporation in Hsp64 is increased severalfold immediately after HS to MTs. However, Western blotting did not show a corresponding increase in the level of Hsp64 after HS, although the levels of Hsp70 were found to change in the same blots in parallel with 35
S-methionine–labeling data. The constant level of Hsp64 in MT cells under different conditions contrasts with the well-known increase in the net cellular levels of the various Hsps after their synthesis is stimulated by stress. A possible explanation for the apparent contradiction between the 35
S-methionine–labeling and Western data could be that the antibodies do not recognize the 35
S-methionine–labeled 64-kDa polypeptide in heat-shocked MTs and, therefore, fail to detect its increased level after HS. However, this possibility is ruled out by an earlier observation (Lakhotia and Singh 1996
) that the 64-kDa polypeptide, immunoprecipitated from 35
S-methionine–labeled heat-shocked MT using the SPA805 antibody, showed as much increased labeling over that from control MT as in total protein samples. Thus, the SPA805 polyclonal antibody recognizes the HS-induced Hsp64. In addition, we have now also used another monoclonal antibody SPA806 from Stressgen, which identifies only a specific epitope of the Hsp64. This particular epitope is fairly well conserved (see ) in the 3 known Hsp64 forms in D melanogaster
(Kozlova et al 1997
; Timakov and Zhang 2001
; Berkeley Drosophila
Genome Project). Thus, this antibody is expected to recognize all forms of the Hsp64 in Drosophila
cells. But it is interesting to note that the SPA806 antibody did not reveal any increase in the level of Hsp64 in heat-shocked MT samples. Thus, we conclude that despite the increased synthesis of Hsp64 in MTs immediately after HS, the net amount of the Hsp64 in these cells does not increase.
Fig. 6. Alignment of the amino acid sequence of the human Hsp60 (accession number X53584) from amino acid residues 383–447 with the 3 Hsp64 protein sequences known in Drosophila melanogaster (Dm10A, Flybase ID number FBgn0015245; Dm21D, Flybase (more ...)
Interestingly, not only did the level of total Hsp64 in MT cells not increase after HS but also when this synthesis was inhibited by actinomycin D or cycloheximide the Hsp64 level in MT remained unaltered. Put together, these results suggest that the new synthesis and turnover of existing Hsp64 are tightly coupled so that the steady-state level of Hsp64 in MT cells remains unaltered. Our finding that newly synthesized (35
S-methionine labeled) Hsp64 disappears within 30 minutes to 60 minutes () also highlights rapid turnover of this protein. A comparable situation that the increased synthesis of a protein is not accompanied by a corresponding increase in the net amount of that protein has been reported earlier for Hsp56 (Sanchez 1990
) and Cyp40 (Mark et al 2001
). Reasons and mechanisms for the regulated level of Hsp64 in MT cells are not clear. Nevertheless, it appears that an increase in Hsp64 in cells, even under stress conditions, may have some unwanted effect, and yet the MT cells may need newly synthesized Hsp64 under conditions of HS. Because actinomycin D treatment during HS prevented 35
S-methionine labeling of Hsp64 in the MT cells, it is clear that the increased synthesis of this protein after HS in MT cells is dependent on new transcription rather than on increased translatability of preexisting transcripts.
Singh and Lakhotia (1995)
demonstrated that the absence of Hsp70 synthesis in larval MTs during or immediately after HS was neither because these tissues already had a basal level of Hsp70 nor because the cognate forms (Hsc70) were heat inducible in these cells. Krebs and Feder (1997)
further confirmed the absence of Hsp70 in larval MTs immediately after HS but showed that the Hsp70 is detectable in these cells during recovery. In this context, the present finding that hsp70
genes at 87A as well as 87C sites are readily induced by HS in larval MTs is intriguing. Present results further showed that unlike in other cells where much of the hsp70 transcripts were degraded within 1–2 hours of recovery, the hsp70
genes in MTs continued to actively transcribe even after 2 hours of recovery from HS and the transcripts persisted till at least 4 hours of recovery. The hsp70 transcripts at this time were most likely not carrying 3′UTR because both the 3′UTR riboprobes failed to detect any transcripts and also the size of the hsp70 transcripts at 4 hours of recovery as seen in the Northern blot was smaller.
The absence of 3′UTR in hsp70 transcripts persisting after 4 hours of recovery is significant because normally hsp70 transcripts are degraded within a short time of recovery as a result of the destabilizing role of the AU-rich elements in 3′UTRs of hsp70 transcripts (Simcox et al 1985
; Yost et al 1990
). Therefore, the absence of 3′UTRs in the hsp70 transcripts present in larval MTs later during recovery may help stabilize the transcripts and allow their translation. Whether the absence of 3′UTR in hsp70 transcripts made during recovery in MT cells is due to a transcriptional or posttranscriptional regulation remains to be examined.
Because the hsp70
genes in larval MTs are quickly induced to transcribe by HS but the protein is not detectable, except in the small stellate cells (Singh and Lakhotia 1995
; our observations), till after some recovery period (Krebs and Feder 1997
; our observations), it is apparent that a posttranscriptional regulation prevents Hsp70 synthesis in these cells. It was seen that compared with other cell types, the hsp70 transcripts in heat-shocked larval MT cells were mostly nuclear and seemed to move to the cytoplasm only during recovery. This delayed transport may be one of the factors that prevents a rapid translation of these mRNAs. Present results further revealed that the delayed appearance of Hsp70 in MT cells was dependent on continuing transcription because inhibition of transcription by actinomycin D during recovery prevented Hsp70 synthesis. It appears therefore that the hsp70 transcripts synthesized during HS are turned over without translation, and the Hsp70 synthesis during recovery depends on the continued transcription of the hsp70
The 2 hsp70-LacZ
reporter transgenic lines shed some light on the possible regulatory events that affect Hsp70 synthesis in MT cells. The rapid expression of HS-induced reporter LacZ
but not in Bg9,
larval MT is significant. Although in most of the larval tissues in the −194a
transgenic line carrying the depleted hsp70
5′UTR the β-galactosidase activity was poorly induced by HS, in the MT cells the reporter activity was easily detectable soon after the HS. Apparently, the difference in the response of the reporter gene to HS in these 2 transgenic lines is related to the deletion of a part (from +85 to +259) of the 5′UTR sequence (see Materials and Methods). Studies by McGarry and Lindquist (1985)
cell lines showed that deletion of sequences from 5′UTRs of hsp70
mRNAs prevented their translation at HS temperature, but these transcripts were translated during recovery (Hess and Duncan 1994
; Duncan 1996
). This may explain the absence of β-galactosidase activity in most tissues of the −194a
larvae immediately after the HS. The same region may also be responsible for the opposing pattern of posttranscriptional and translational regulation of the hsp70 mRNA in MT. In MT cells, the presence of this region in the mRNA (as in those transcribed by the resident hsp70
genes or by the transgene in Bg9
) seems to prevent translation (and possibly transport to the cytoplasm) during or immediately after HS but in its absence (as in the −194a
transgenic larvae) this translational delay does not occur. Present results thus show that the 5′UTR in the hsp70 mRNAs has important regulatory elements, which in addition to being parts of the core promoter (Wu et al 2001
) also have additional posttranscriptional regulatory functions. These need to be identified.
The regulatory mechanisms that cause the transcriptional activation of Hsp64 in MT cells and maintain the steady-state levels of Hsp64 in MT cells under all conditions remain to be understood. The significance of this unusual HS response is also not clear. However, because MTs in several other insects also show varying degrees of variations from the basal HS response (Nath and Lakhotia 1989
; Tiwari et al 1995
; Singh and Lakhotia 2000
), it is likely that the specific cellular physiology of MT cells has some specific requirements.
Another recent study from our laboratory (Lakhotia and Prasanth 2002
) has shown that there are cell- and developmental stage–specific variations in the HS-induced transcription of the different hsp70
genes of D melanogaster
and turnover of their transcripts. The present studies reveal another example of the fine-tuning of the HS response in relation to specific cellular requirements. Apparently, we still have much to learn about the finer details of an individual cell's response to stress.