Finding a niche

Although I always knew I wanted to be a scientist, I didn't know I would become a cell biologist. Events in life that you would never have predicted can greatly impact your career trajectory. I have learned to let those events take me in new directions. Following a desire to investigate an understudied area of cell biology, I have found a niche. In this area, my lab is poised to contribute significantly toward understanding the fundamental molecular mechanisms underlying polarized plant cell growth.     

Characterization of a new high-temperature-induced 66-kDa heat-shock protein,antigenically related to heat-shock protein 72

M-14 human melanoma cells, following severe hyperthermic exposures, synthesized a heat-shock protein of 66 kDa (hsp 66), in addition to the major “classic” heat-shock proteins. This hsp 66 was not expressed following mild hyperthermic exposures sufficient to trigger the synthesis of the other heat-shock proteins. The induction of hsp 66 was observed also in Li human glioma cells treated at 45°C for 20 min. By contrast, hsp 66 was not induced in seven other human cell lines (both melanoma and nonmelanoma) when they were subjected to the same hyperthermic treatment. Immunological recognition experiments showed that hsp 66 cross-reacted with the inducible hsp 72, but not with the constitutive hsp 73. The possibility that hsp 66 is a breakdown product of hsp 72 was ruled out by the fact that Poly(A)⁺ RNA extracted from cells treated at 45°C for 20 min was able to direct the synthesis of hsp 66 (together with hsp 72) in a message-dependent rabbit reticulocyte lysate, as well as in microinjected Xenopus oocytes. By contrast, only the hsp 72 was expressed using Poly(A)⁺ RNA extracted from cells heated at 42°C for 1 h. Affinity chromatography experiments on ATP-agarose showed that hsp 66 did not bind ATP in vitro, hsp 66 was localized both in the cytoplasm (cytosol, mitochondria, and microsome fraction) and in the nuclei of cells recovered from a severe heat shock: this intracellular distribution closely corresponded to that of hsp 72. The nuclear-associated hsp 66 was found to be tightly bound to nuclear structures and could not be extracted by incubation in ATP-containing buffer. © 1996 Wiley-Liss, Inc.     

The foot as a shock absorber

A mathematical analysis of the deformation of the foot is developed to determine the role that stretch of ligaments and tendons plays in absorbing shock following impact. Our analysis is based on an anatomical biomechanical model that includes each of the bones of the foot. We calculate the time course of the deflection of the joints and the elongation of the ligaments and tendons and determine the ground reaction force acting on the heel. Quasi-linear viscoelastic theory is used for soft tissue constitutive relationships. With biomechanical data selected from the literature, we obtain a vertical force impact peak of 8000 N, occurring at 16 ms following heel strike. This is of higher magnitude and shorter duration than is found experimentally, as is to be expected, since we did not include the heel pad in our model and we assumed that the impact surface was ideally rigid.     

Evidence for protection by heat-shock proteins against photoinhibition during heat-shock

The nuclear-coded 22 kd heat-shock protein (HSP-22) which is transported into the chloroplast and localized in the thylakoids was further characterized and found to be located in the grana lamellae (stacked thylakoids) as an extrinsic protein in the green alga Chlamydomonas reinhardtii. Inhibition of photosynthetic electron flow during heat-shock of Chlamydomonas cells was light-dependent, occurring at low-light intensities (<100 W/m²) as compared with photoinhibition at 25°C (>1000 W/m²). The site of the damage was localized at the photosystem II (PS II) reaction center. The damage was drastically increased when heat-shock treatment was carried out in the presence of the 80S ribosomal translation inhibitor, cycloheximide (CHI). Pre-incubation of Chlamydomonas cells at 42°C resulted in partial protection against photoinhibition during heat-shock, as compared with cells pre-incubated at 42°C in the presence of CHI which, therefore, did not translate the heat-shock proteins. Analysis of the thylakoid polypeptides' pattern by SDS-PAGE revealed that during heat-shock in the light, thylakoid proteins became aggregated proportionally to the light intensity. Heat-shock in the presence of CHI enhanced the aggregation process which, at low light intensities, was specific to the PS II reaction center D1-protein. The results suggest that the chloroplasts HSPs prevent damage to the PS II reaction center during heat-shock in the light.     

Archaebacterial heat-shock proteins

The response to heat shock was examined in seven archaebacterial strains from the genus Halobacterium. Upon heat shock each strain preferentially synthesized a limited number of proteins which fell into three narrow mol. wt. ranges. Further examination of the heat-shock response in H. volcanii revealed that heat-shock protein (hsp) synthesis was greatest at 60°C. Synthesis of hsps at this induction temperature was both rapid and transient. Cells recovered their normal protein synthesis patterns rapidly upon returning to their normal growth temperature following heat shock. H. volcanii cells also responded with a `heat shock-like' response to salt dilution, a natural environmental stress for these organisms. These results indicate that the heat shock or stress response which is charactertistic of eukaryotic and eubacterial cells is also present among members of the archaebacterial genus Halobacterium.     

Finding a ribophagy receptor

Although ribophagy was demonstrated in budding yeast a decade ago, a specific receptor for this process has been unknown. Recently, a study revealed that NUFIP1 (nuclear FMR1 interacting protein 1) functions as a receptor for the selective degradation of ribosomes by starvation-induced autophagy in cultured mammalian cells. In addition to the identification of a selective autophagy receptor, this study suggests a strategy that can be adapted to the identification of additional novel receptor proteins.     

Parasite heat-shock proteins

Many parasites, including most of those of medical or veterinary importance, experience a major increase in ambient temperature at some stage during their life cycle. This occurs when a cyst or free-living larval form is ingested by a warm-blooded host, when a poikilotherm-infecting parasite is transmitted to a homeotherm, or when a transiently free-living invasive larva penetrates the skin of a mammal. This sudden change in temperature could be expected to stress the intruder, as it should dramatically alter rates of metabolic reactions and of denaturation of proteins. This would especially affect the function of near-equilibrium, regulatory, and membrane-bound enzymes (changes in temperature affect membrane fluidity). In this article George Newport, Janice Culpepper and Nina Agabian consider how parasites cope with this problem, emphasizing the possible role of heat-shock proteins (HSPs), how the expression of these molecules is regulate, and how HSPs interact with the host immune system.     

Identification of a heat-shock pseudogene from Caenorhabditis elegans

While characterizing the hsp70 gene family from Caenorhabditis elegans we encountered an unusual member of this family. Sequence data reveal that the hsp-2ps gene is a pseudogene of the constitutively expressed, heat-inducible hsp-1 gene. Two stop codons generated near the 5' end of the sequence as well as several frameshift mutations and a large internal deletion confirm the identification of hsp-2ps as a pseudogene. The nucleotide substitution rate of the third codon position was twice that of the first and second codon positions, suggesting that the hsp-2ps gene was nonfunctional since the time of the duplication event. The hsp-2ps gene duplicates a region of the hsp-1 gene that lies exclusively within the transcribed region and retains the introns. We feel that the hsp-2ps gene was produced by a transpositional duplication event, which occurred approximately 8.5 million years ago.     

Induction of a proteinase by heat-shock in yeast

Abstract In Saccharomyces cerevisiae heat-shock induces an increase in proteinase activity. The induction is probably due to newly synthesized enzyme molecules, since the increase in proteinase activity can be inhibited by cycloheximide. Degradation of endogenous proteins is enhanced by EDTA, while the azocasein assay is not affected by MnCl₂, MgCl₂, or EDTA. The proteinase has a pH optimum of 8, and phenylmethylsulfonyl fluoride (PMSF) as well as chymostatin are strong inhibitors. We infer that the induced proteinase is probably identical with proteinase B of yeast.     

How to build a molecular shock absorber

Newly determined structures of the alpha-helical repeats that make up the key 'rod' domains of spectrin and alpha-actinin - which serve as spacers between their actin-binding domains - have provided important insights into how these proteins function as molecular shock absorbers in cells.     

Finding a target for resveratrol

Despite resveratrol's well-documented health benefits, its mechanism of action remains controversial. In particular, the direct molecular target of resveratrol has been elusive. Park et?al. now show that resveratrol directly inhibits cAMP-dependent phosphodiesterases, triggering a cascade of events that converge on the important energy-sensing metabolic regulators AMPK, SIRT1, and PGC-1α.     

Clustering of heat-shock factors

Clusterin is a ubiquitous glycoprotein found in most physiological fluids and tissues. Although not fully understood, the function of clusterin seems to be related to its ability to bind a wide variety of molecules. Since clusterin has been found associated with extracellular protein aggregates, a role as a molecular chaperone has been proposed. In this issue of the Biochemical Journal, Le Dréan and colleagues demonstrate an up-regulation of clusterin in neuronal cells exposed to proteotoxic stress that results in unfolded protein accumulation and proteasome impairment, both commonly associated with neurodegenerative diseases. Interestingly, expression of clusterin was found to be regulated by two members of the HSF (heat-shock factor) family, HSF1 and HSF2, which possibly form a trimeric complex on the clusterin promoter. The study proposes clusterin as a player in a cellular defence mechanism against harmful protein accumulation, and highlights the importance of elucidating further the exact role of clusterin and the intriguing interaction between HSF1 and HSF2.