S. C. Lakhotia

Heat shock genes - integrating cell survival and death.

Heat shock induced gene expression and other cellular responses help limit the damage caused by stress and thus facilitate cellular recovery. Cellular damage also triggers apoptotic cell death through several pathways. This paper briefly reviews interactions of the major heat shock proteins with components of the apoptotic pathways. Hsp90, which acts as a chaperone for unstable signal transducers to keep them poised for activation, interacts with RIP and Akt and promotes NF-κB mediated inhibition of apoptosis; in addition it also blocks some steps in the apoptotic pathways. Hsp70 is mostly anti-apoptotic and acts at several levels like inhibition of translocation of Bax into mitochondria, release of cytochrome c from mitochondria, formation of apoptosome and inhibition of activation of initiator caspases. Hsp70 also modulates JNK, NF-κB and Akt signaling pathways in the apoptotic cascade. In contrast, Hsp60 has both anti-and pro-apoptotic roles. Cytosolic Hsp60 prevents translocation of the pro-apoptotic protein Bax into mitochondria and thus promotes cell survival but it also promotes maturation of procaspase-3, essential for caspase mediated cell death. Our recent in vivo studies show that RNAi for the Hsp60D in Drosophila melanogaster prevents induced apoptosis. Hsp27 exerts its anti-apoptotic influence by inhibiting cytochrome c and TNF-mediated cell death. αβ crystallin suppresses caspase-8 and cytochrome c mediated activation of caspase-3. Studies in our laboratory also reveal that absence or reduced levels of the developmentally active as well as stress induced non-coding hsrω transcripts, which are known to sequester diverse hnRNPs and related nuclear RNA-binding proteins, block induced apoptosis in Drosophila. Modulation of the apoptotic pathways by Hsps reflects their roles as “weak links” between various “hubs” in cellular networks. On the other hand, non-coding RNAs, by virtue of their potential to bind with multiple proteins, can act as “hubs” in these networks. In view of the integrative nature of living systems, it is not surprising that stress-induced genes, generally believed to primarily function in cell survival pathways, inhibit or even promote cell death pathways at multiple levels to ensure homeostasis at cell and/or organism level. The heat shock genes obviously do much more than merely help cells survive stress.

Specific activation of puff 93D of Drosophila melanogaster by benzamide and the effect of benzamide treatment on the heat shock induced puffing activity

A 10 or 20 min in vitro treatment of salivary glands of late 3rd instar larvae of Drosophila melanogaster with l mg/ml benzamide (BM) at 24° C results in the specific induction of the 93 D puff and at the same time all other chromosomal RNA synthesis is severely repressed. Incorporation of 3H-uridine in the nucleolus is not affected. In glands heat shocked (37° C) for 20 min in presence of BM, all the temperature shock (TS) puffs are induced but they incorporate 3H-uridine to a lesser extent than in glands heat shocked in absence of BM. The 93 D puff, which is highly induced by either of the treatments alone, is relatively less active in glands exposed to TS and BM simultaneously. When a 10 min BM treatment (at 24° C) precedes a 20 min TS or when the BM treatment follows TS, the 3H-uridine incorporation on all TS puffs is relatively less and significantly, in both cases, the 87 C puff is much less active (more so in TS followed by BM treated glands) than the 87 A puff. Also, in both these treatments, the 93 D puff does not show any additive effect of the BM and TS treatments. These observations are discussed in the light of possible role of the 93 D puff in modulating the heat shock response.

Chromosomal basis of dosage compensation in Drosophila: I. Cellular autonomy of hyperactivity of the male X-chromosome in salivary glands and sex differentiation

Morphology and the rate of RNA synthesis of the X -chromosome in XX/XO mosaic larval salivary glands of Drosophila melanogaster have been examined. For this purpose the unstable ring- X was utilized to produce XX and XO nuclei in the same pair of glands. The width of the X -chromosome and the left arm of the 3rd chromosome (3 L ) of larval salivary glands was measured and the rate of RNA synthesis by them was studied upon the use of [ 3 H]uridine autoradiography in such XX (female) and XO (male) nuclei developing in a female background (i.e. otherwise genotypically XX ). In such mosaic glands the width of the single X -chromosome of male nuclei is nearly as great as that of the paired two X s of female nuclei, as is also the case in normal male ( X Y ) and female ( XX ). The single X of male nuclei synthesizes RNA at a rate equal to that of the paired two X s of female nuclei and nearly twice that of an unpaired X of XX nuclei. Neither the developmental physiology of the sex nor the proportion of XO nuclei in a pair of mosaic salivary glands of an XX larva has any influence on these two characteristics of the male X -chromosome. It is suggested that dosage compensation in Drosophila is achieved chiefly, if not fully, by a hyperactivity of the male X , in contrast to the single X inactivation in female mammals, that this hyperactivity of the male X is expressed visibly in the morphology and metabolic activity of the X -chromosome in the larval salivary glands of the male, and that this hyperactivity and therefore dosage compensation in Drosophila in general is not dependent on sex-differentiation, but is a function of the doses of the X -chromosome itself.

EM autoradiographic studies on polytene nuclei of Drosophila melanogaster: II. Organization and transcriptive activity of the chromocentre

Abstract The organization and the transcriptive activity of the chromocentre region of salivary gland nuclei of Drosophila melanogaster have been examined by electron microscopy and EM autoradiography. The heterochromatic chromocentre region is organized into a central compact block of chromatin, surrounded by a large number of smaller, interconnected blocks. The latter are continued into the euchromatic chromosome arms. The two types of chromatin organization in the chromocentre are reminiscent of the classical α- and β-heterochromatin. Two types of ribonucleoprotein-like particles regularly occur in the β-heterochromatin. EM autoradiography after short (5 min) in vitro pulse of 3 H-uridine shows that the β-heterochromatin is as active in RNA synthesis as the euchromatin in the nucleus; the α-heterochromatin is completely inactive. It is suggested that the two types of particles seen in the β-heterochromatin are products of RNA synthesis occurring in this region. These observations raise doubts on the general validity of the notion of total inactivity of constitutive heterochromatin.

In Vivo Effects Of Traditional Ayurvedic Formulations in Drosophila melanogaster Model Relate with Therapeutic Applications

Background Ayurveda represents the traditional medicine system of India. Since mechanistic details of therapy in terms of current biology are not available in Ayurvedic literature, modern scientific studies are necessary to understand its major concepts and procedures. It is necessary to examine effects of the whole Ayurvedic formulations rather than their “active” components as is done in most current studies. Methods We tested two different categories of formulations, a Rasayana (Amalaki Rasayana or AR, an herbal derivative) and a Bhasma (Rasa-Sindoor or RS, an organo-metallic derivative of mercury), for effects on longevity, development, fecundity, stress-tolerance, and heterogeneous nuclear ribonucleoprotein (hnRNP) levels of Drosophila melanogaster using at least 200 larvae or flies for each assay. Results A 0.5% (weight/volume) supplement of AR or RS affected life-history and other physiological traits in distinct ways. While the size of salivary glands, hnRNP levels in larval tissues, and thermotolerance of larvae/adult flies improved significantly following feeding either of the two formulations, the median life span and starvation resistance improved only with AR. Feeding on AR or RS supplemented food improved fecundity differently. Feeding of larvae and adults with AR increased the fecundity while the same with RS had opposite effect. On the contrary, feeding larvae on normal food and adults on AR supplement had no effect on fecundity but a comparable regime of feeding on RS-supplemented food improved fecundity. RS feeding did not cause heavy metal toxicity. Conclusions The present study with two Ayurvedic formulations reveals formulation-specific effects on several parameters of the flys life, which seem to generally agree with their recommended human usages in Ayurvedic practices. Thus, Drosophila, with its very rich genetic tools and well-worked-out developmental pathways promises to be a very good model for examining the cellular and molecular bases of the effects of different Ayurvedic formulations.

Long non-coding RNAs coordinate cellular responses to stress.

Following the initial discovery of the heat shock RNA omega (hsrω) gene of Drosophila melanogaster to be non‐coding (nc) and also inducible by cell stress, other stress‐inducible long non‐coding RNAs (lncRNA) have been described in diverse organisms. In view of the rapid sequence divergence of lncRNAs, present knowledge of stress trasncriptome is limited and fragmented. Several known stress‐related lncRNAs, associated with specific nuclear speckled domains or nucleolus, provide structural base for sequestering diverse RNA‐processing/regulatory proteins. Others have roles in transcriptional or translational inhibition during stress or in signaling pathways; functions of several other lncRNAs are not yet known. Most stress‐related lncRNAs act primarily by modulating activity of the proteins to which they bind or by sequestering specific sets of proteins away from the active pool. A common emerging theme is that a given lncRNA targets one or more protein/s with key role/s in the cascade of events triggered by the stress and therefore has a widespread integrative effect. Since proteins associate with RNA through short sequence motifs, the overall base sequence of functionally similar ncRNAs is often not conserved except for specific motifs. The rapid evolvability of ncRNA sequences provides elegant modules for adaptability to changing environment as binding of one or the other protein to ncRNA can alter its structure and functions in distinct ways. Thus the stress‐related lncRNAs act as hubs in the cellular networks to coordinate activities of the members within and between different networks to maintain cellular homeostasis for survival or to trigger cell death. WIREs RNA 2012. doi: 10.1002/wrna.1135

The large noncoding hsrω-n transcripts are essential for thermotolerance and remobilization of hnRNPs, HP1 and RNA polymerase II during recovery from heat shock in Drosophila.

The hs-GAL4t-driven expression of the hsrω-RNAi transgene or EP93D allele of the noncoding hsrω resulted in global down- or upregulation, respectively, of the large hsrω-n transcripts following heat shock. Subsequent to temperature shock, hsrω-null or those expressing hsrω-RNAi or the EP93D allele displayed delayed lethality of most embryos, first or third instar larvae. Three-day-old hsrω-null flies mostly died immediately or within a day after heat shock. Heat-shock-induced RNAi or EP expression in flies caused only a marginal lethality but severely affected oogenesis. EP allele or hsrω-RNAi expression after heat shock did not affect heat shock puffs and Hsp70 synthesis. Both down- and upregulation of hsrω-n transcripts suppressed reappearance of the hsrω-n transcript-dependent nucleoplasmic omega speckles during recovery from heat shock. Hrp36, heterochromatin protein 1, and active RNA pol II in unstressed or heat-shocked wild-type or hsrω-null larvae or those expressing the hs-GAL4t-driven hsrω-RNAi or the EP93D allele were comparably distributed on polytene chromosomes. Redistribution of these proteins to pre-stress locations after a 1- or 2-h recovery was severely compromised in glands with down- or upregulated levels of hsrω-n transcripts after heat shock. The hsrω-null unstressed cells always lacked omega speckles and little Hrp36 moved to any chromosome region following heat shock, and its relocation to chromosome regions during recovery was also incomplete. This present study reveals for the first time that the spatial restoration of key regulatory factors like hnRNPs, HP1, or RNA pol II to their pre-stress nuclear targets in cells recovering from thermal stress is dependent upon critical level of the large hsrω-n noncoding RNA. In the absence of their relocation to pre-stress chromosome sites, normal developmental gene activity fails to be restored, which finally results in delayed organismal death.

Expression of mdr49 and mdr65 multidrug resistance genes in larval tissues of Drosophila melanogaster under normal and stress conditions

Abstract In situ expression of 2 multidrug resistance genes, mdr49 and mdr65, of Drosophila melanogaster was examined in wild-type third instar larval tissues under physiological conditions and after heat shock or colchicine feeding. Expression of these 2 genes was also examined in tumorous tissues of lethal (2) giant larvae l(2)gl4 mutant larvae. These 2 mdr genes show similar constitutive expression in different larval tissues under physiological conditions. However, they are induced differentially by endogenous (tumorous growth) and exogenous stresses (colchcine feeding or heat shock): whereas heat shock and colchicine feeding induce mdr49, tumorous condition is accompanied by enhanced expression of mdr49 and mdr65 genes.

Tissue-specific variations in the induction of Hsp70 and Hsp64 by heat shock in insects

Abstract Abstract The patterns of heat-induced synthesis (37°C to 45°C) of heat shock proteins (Hsps) in different tissues of grasshoppers and cockroaches from natural populations and in laboratory-reared gram-pest (Heliothis armigera) were examined by 35S-methionine labeling and sodium dodecyl sulfate–polyacrylamide gel electrophoresis fluorography. Whereas 45°C was lethal in most cases, optimal induction of Hsp synthesis was seen between 37°C and 42°C. The ongoing protein synthesis was not much affected at these temperatures, except in the tissues of adult H armigera exposed to 42°C. The profiles of the Hsps induced in the tissues of the insects, however, were different. From the relative abundance of the synthesis of 70-kDa (Hsp70) and 64-kDa (Hsp64) polypeptides, three categories of heat shock response were identified: (1) induction of abundant Hsp70 but little Hsp64 (malpighian tubules, male accessory glands, and ovaries of adult grasshoppers), (2) abundant Hsp64 but little Hsp70 (testes of adult grasshoppers, testes and malpighian tubules of adult cockroaches, and testes, malpighian tubules, and fat bodies of H armigera larvae), and (3) induction of both Hsp70 and Hsp64 in more or less equal abundance (ovaries of adult cockroaches, salivary glands of H armigera larvae, and malpighian tubules, male accessory glands, testes, and ovaries of adult H armigera). Cockroaches collected from storerooms showed detectable synthesis of Hsp64 and/or Hsp70 only after heat shock, but those collected from drains showed detectable synthesis of both Hsp70 and Hsp64 in different tissues without heat stress. Western blotting showed that the 64-kDa polypeptide in these insects is a member of the Hsp60 family. Grasshopper testes, which synthesized negligible Hsp70 but abundant Hsp64 after heat shock, developed thermotolerance. Thus, heat shock response is modulated by developmental and environmental factors in different tissues of insects.

Absence of novel translation products in relation to induced activity of the 93D puff in Drosophila melanogaster

Salivary glands of Drosophila larvae were treated in vitro with benzamide or with a homogenate of heat shocked glands to specifically induce high transcriptional activity of the 93D puff. The newly synthesized 14C-amino acids labelled polypeptides in the treated and sister control glands were analysed by polyacrylamide gel electrophoresis, followed by gel autoradiography. The protein synthesis patterns in the treated glands in either case remain the same as in control glands. No novel polypeptide was seen which could be correlated with the high induced transcriptional activity of the 93D puff. This suggests that the 93D transcript/s is/are probably not translated.