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

In heat-shocked tomato cell cultures, cytoplasmic heat shock granules (HSGs) are tightly associated with a specific subset of mRNAs coding mainly for the untranslated control proteins. This messenger ribonucleoprotein complex was banded in a CsCl gradient after fixation with formaldehyde (approximately 1.30 g/cm3). It contains all the heat shock proteins and most of the RNA applied to the gradient. During heat shock, a reversible aggregation of HSGs from 15S precursor particles can be shown. These pre-HSGs are not identical to the 19S plant prosomes. Ultrastructural analysis supports the ribonucleoprotein nature of HSGs and their composition of approximately 10-nm precursor particles. A model summarizes our results. It gives a reasonable explanation for the striking conservation of untranslated mRNAs during heat shock and may apply also to animal cells.

Formation of cytoplasmic heat shock granules in tomato cell cultures and leaves.

Biochemical and electron microscopic analyses of heat-shocked suspension cultures of Peruvian tomato (Lycopersicon peruvianum) revealed that a considerable part of the dominant small heat shock proteins (hsps) with an Mr of approximately 17,000 are structural proteins of newly forming granular aggregates in the cytoplasm (heat shock granules), whose formation strictly depends on heat shock conditions (37 to 40 degrees C) and the presence or simultaneous synthesis of hsps. However, under certain conditions, e.g., in preinduced cultures maintained at 25 degrees C, hsps also accumulate as soluble proteins without concomitant assembly of heat shock granules. Similar heat shock-induced cytoplasmic aggregates were also observed in other cell cultures and heat-shocked tomato leaves and corn coleoptiles.

The balance of nuclear import and export determines the intracellular distribution and function of tomato heat stress transcription factor HsfA2.

Tomato heat stress transcription factor HsfA2 is a shuttling protein with dominant cytoplasmic localization as a result of a nuclear import combined with an efficient export. Besides the nuclear localization signal (NLS) adjacent to the oligomerization domain, a C-terminal leucine-rich motif functions as a nuclear export signal (NES). Mutant forms of HsfA2 with a defective or an absent NES are nuclear proteins. The same is true for the wild-type HsfA2 if coexpressed with HsfA1 or in the presence of export inhibitor leptomycin B (LMB). Fusion of the NES domain of HsfA2 to HsfB1, which is a nuclear protein, caused export of the HsfB1-A2NES hybrid protein, and this effect was reversed by the addition of LMB. Due to the lack of background problems, Chinese hamster ovary (CHO) cells represent an excellent system for expression and functional analysis of tomato Hsfs. The results faithfully reflect the situation found in plant cells (tobacco protoplasts). The intriguing role of NLS and NES accessibility for the intracellular distribution of HsfA2 is underlined by the results of heat stress treatments of CHO cells (41 degrees C). Despite the fact that nuclear import and export are not markedly affected, HsfA2 remains completely cytoplasmic at 41 degrees C even in the presence of LMB. The temperature-dependent conformational transition of HsfA2 with shielding of the NLS evidently needs intramolecular interaction between the internal HR-A/B and the C-terminal HR-C regions. It is not observed with the HR oligomerization domain (HR-A/B region) deletion form of HsfA2 or in HsfA2-HsfA1 hetero-oligomers.

The tomato Hsf system: HsfA2 needs interaction with HsfA1 for efficient nuclear import and may be localized in cytoplasmic heat stress granules.

In heat-stressed (HS) tomato (Lycopersicon peruvianum) cell cultures, the constitutively expressed HS transcription factor HsfA1 is complemented by two HS-inducible forms, HsfA2 and HsfB1. Because of its stability, HsfA2 accumulates to fairly high levels in the course of a prolonged HS and recovery regimen. Using immunofluorescence and cell fractionation experiments, we identified three states of HsfA2: (i) a soluble, cytoplasmic form in preinduced cultures maintained at 25 degrees C, (ii) a salt-resistant, nuclear form found in HS cells, and (iii) a stored form of HsfA2 in cytoplasmic HS granules. The efficient nuclear transport of HsfA2 evidently requires interaction with HsfA1. When expressed in tobacco protoplasts by use of a transient-expression system, HsfA2 is mainly retained in the cytoplasm unless it is coexpressed with HsfA1. The essential parts for the interaction and nuclear cotransport of the two Hsfs are the homologous oligomerization domain (HR-A/B region of the A-type Hsfs) and functional nuclear localization signal motifs of both partners. Direct physical interaction of the two Hsfs with formation of relatively stabile hetero-oligomers was shown by a two-hybrid test in Saccharomyces cerevisiae as well as by coimmunoprecipitation using tomato and tobacco whole-cell lysates.

External rotation of the femoral component increases asymmetry of the posterior condyles.

AIMS: The morphometry of the distal femur was largely studied to improve bone-implant fit in total knee arthroplasty (TKA), but little is known about the asymmetry of the posterior condyles. This study aimed to investigate the dimensions of the posterior condyles and the influence of externally rotating the femoral component on potential prosthetic overhang or under-coverage. PATIENTS AND METHODS: We analysed the shape of 110 arthritic knees at the time of primary TKA using pre-operative CT scans. The height and width of each condyle were measured at the posterior femoral cut in neutral position, and in 3º and 5º of external rotation, using both central and medial referencing systems. We compared the morphological characteristics with those of 14 TKA models. RESULTS: In the neutral position, the dimensions of the condyles were nearly equal. Externally rotating the femoral cut by 3º and 5º with 'central referencing' induced width asymmetry > 3 mm in 23 (21%) and 33 (30%) knees respectively, while with 'medial referencing' it induced width asymmetry > 3 mm in 43 (39%) and 75 (68%) knees respectively. The asymmetries induced by rotations were not associated with gender, aetiology or varus-valgus alignment. CONCLUSION: External rotation may amplify the asymmetry between the medial and lateral condyles, and exacerbate prosthetic overhang, particularly in the superolateral zone. 'Central referencing' guides result in less potential prosthetic overhang than 'medial referencing' guides. Cite this article: Bone Joint J 2017;99-B:894-903.

Channelling of exogenous phenylalanine to the sites of storage and the sites of alkaloid and protein biosynthesis in Penicillium cyclopium.

Externally applied L-phenylalanine rapidly equilibrates with the cytosolic pool(s) in hyphae of emerged cultures of Penicillium cyclopium. If not incorporated into protein it is accumulated in the so called expandable pool, which is presumably localized in the vacuolar compartment. At high concentrations of exogenous L-phenylalanine practically all of the amino acid needed for protein synthesis comes from the extracellular source, contrary to alkaloid synthesis which under all conditions recruits more than 90% of the required L-phenylalanine from intracellular sources. Two pathways of alkaloid labelling can be distinguished, by which externally applied L-phenylalanine reaches the sites of alkaloid synthesis: (1) a direct way from cytosolic pool (primary labelling) and (2) an indirect way via the expandable pool (secondary labelling).

Heat shock induced changes of plant cell ultrastructure and autoradiographic localization of heat shock proteins.

Treating tomato cell cultures and leaves by a physiological heat shock (hs) at 35 to 39 degrees C results in a progressive disintegration of the nucleolus and the assembly of cytoplasmic hs granules. Other ultrastructural changes are not observed. The alterations of the nucleoli coincide with an immediate stop of the processing and with a strongly decreased synthesis of pre-rRNA. Both hs effects are reversed after shift-down to normal temperature conditions (25 degrees C). Assembly of cytoplasmic hs granules depends on the accumulation of the newly forming hs proteins and on supraoptimal temperatures. It is not observed in preinduced cultures synthesizing hs proteins at 25 degrees C. Autoradiographic studies reveal the preferential accumulation of hsp in the nucleoli and hs granules. Furthermore uridine labeling points to the presence of RNA in electron dense particles of both subcellular components. A survey on the state of hsp synthesis and structural binding as well as on the ultrastructural changes is given for 12 selected hs regimes.

Arabidopsis and the heat stress transcription factor world: how many heat stress transcription factors do we need?

Sequencing of the Arabidopsis genome revealed a unique complexity of the plant heat stress transcription factor (Hsf) family. By structural characteristics and phylogenetic comparison, the 21 representatives are assigned to 3 classes and 14 groups. Particularly striking is the finding of a new class of Hsfs (AtHsfC1) closely related to Hsf1 from rice and to Hsfs identified from frequently found expressed sequence tags of tomato, potato, barley, and soybean. Evidently, this new type of Hsf is well expressed in different plant tissues. Besides the DNA binding and oligomerization domains (HR-A/B region), we identified other functional modules of Arabidopsis Hsfs by sequence comparison with the well-characterized tomato Hsfs. These are putative motifs for nuclear import and export and transcriptional activation (AHA motifs). There is intriguing flexibility of size and sequence in certain parts of the otherwise strongly conserved N-terminal half of these Hsfs. We have speculated about possible exon-intron borders in this region in the ancient precursor gene of plant Hsfs, similar to the exon-intron structure of the present mammalian Hsf-encoding genes.

The Hsf world: classification and properties of plant heat stress transcription factors.

Based on the partial or complete sequences of 14 plant heat stress transcription factors (Hsfs) from tomato, soybean, Arabidopsis and maize we propose a general nomenclature with two basic classes, i.e. classes A and B each containing two or more types of Hsfs (HsfA1, HsfA2 etc.). Despite some plant-specific peculiarities, essential functional domains and modules of these proteins are conserved among plants, yeast, Drosophila and vertebrates. A revised terminology of these parts follows recommendations agreed upon among the authors and representatives from other laboratories working in this field (see legend to Fig. 1). Similar to the situation with the small heat shock proteins (sHsps), the complexity of the hsf gene family in plants appears to be higher than in other eukaryotic organisms.

[Molecular cell biology of the heat stress response. Part I]. / Molekulare Zellbiologie der Hitzestressantwort. Teil I.

In a physiological range of hyperthermia all living systems respond with a complex reprogramming of cellular activities to provide a basis for survival during the stress period and for a rapid restoration of normal activities in the recovery period. A prominent characteristic of the response is the induced synthesis of heat-stress proteins which is likewise evoked by numerous chemical stressors. The common signal transduction chain leading to the activation of heat-stress genes evidently involves the transient accumulation of abnormal proteins. The dominant HSPs belong to five conserved stress protein families, whose members are essential components of all living cells with general functions by far exceeding the stress response.

125 years of experimental heat shock research: historical roots of a discipline.

The history of experimental heat shock research over the last 125 years is briefly, outlined. Starting with reports on the upper temperature limits of plant survival (1864) and on the spontaneous regression of skin cancer after a severe local inflammation (1866), studies on the heat shock response today are a major field of modern cell biology and include all types of prokaryotic and eukaryotic organisms.

[Molecular cell biology of the heat stress response. II]. / Molekulare Zellbiologie der Hitzestressantwort. Teil II.

The coordinate induction of distinct genes by heat stress and a considerable number of chemical stressors depend on a common regulatory element in the promoter found in front of related genes of all eukaryotic systems. This element can be used for the construction of universal heat stress expression cassettes. The increasingly broad interest in the heat stress response and the genes involved also results from medical aspects, e.g., the potential application of hyperthermia in cancer therapy, the intricate connections of stress proteins and genes with malignant transformation, and the remarkable role of heat stress proteins as dominant antigens of infectious and autoimmune diseases.

Stable transformation of an Arabidopsis cell suspension culture with firefly luciferase providing a cellular system for analysis of chaperone activity in vivo.

Using Agrobacterium, we developed a method to transform an Arabidopsis cell suspension culture. A stably transformed cell line expressing high levels of firefly luciferase (Luc) was used for in vivo studies of thermal denaturation and renaturation of the enzyme and the protective role of different chaperones. Luc activity was monitored under heat stress and recovery conditions in control, thermotolerant cells and cells expressing plant chaperones after transient cotransformation with plasmids encoding proteins of the heat shock protein Hsp90, Hsp70, or Hsp20 family. The effects of the expressed proteins were specific. The Hsp17.6 class I protein maintained Luc activity on a level comparable with that observed in thermotolerant cells and improved Luc renaturation. Although transient expression of Hsp90 did not protect Luc from thermal denaturation, it accelerated Luc renaturation during recovery. In contrast to the other chaperones tested, overexpression of Hsp70 alone had no effect on denaturation and renaturation of Luc but enhanced Luc renaturation if coexpressed with Hsp17.6.

Heat-shock-induced alterations of ribosomal protein phosphorylation in plant cell cultures.

Heat shock of cell suspension cultures of tomato (Lycopersicon peruvianum) results in a rapid and reversible decline of the phosphorylation level of a single basic ribosomal protein of the small subunit (tentatively identified as ribosomal protein S6). Simultaneously, phosphate labeling of several acidic ribosomal proteins of the large subunit is enhanced. Data on the temperature-dependent distribution of S6 subspecies and on the kinetics and reversibility of S6 phosphorylation are given. The decreased phosphorylation of S6 at temperatures higher than 35 degrees C coincides with the onset of heat shock protein synthesis and precedes a decline of the mitotic index. Recovery from heat shock is characterized by S6 rephosphorylation and, subsequently, leads to an abnormally high mitotic index.

Synthesis, modification and structural binding of heat-shock proteins in tomato cell cultures.

Synthesis of about 30 acidic and 18 basic heat-shock proteins (hsps) is induced in suspension cultures of tomato (Lycopersicon peruvianum) if subjected to supraoptimal temperature conditions (35-40 degrees C). A characteristic aspect of the plant heat-shock response is the formation of cytoplasmic granular aggregates, heat-shock granules, containing distinct heat-shock proteins as major structural components and, in addition, several hitherto undetected minor acidic and basic heat-shock proteins. Structural binding of heat-shock proteins, i.e. assembly of heat-shock granules, is dependent on the persistance of supraoptimal temperature conditions. Despite the ongoing synthesis also at 25 degrees C, e.g. in pulse heat-shocked cultures, these proteins are accumulated exclusively in soluble form. Individual heat-shock proteins are characterized by their kinetics of synthesis and are classified by their compartmentation behaviour into class A proteins (exclusively found in soluble form, e.g. hsps 95 and 80), class B proteins (5-10% bound to heat-shock granules, e.g. hsps 70, 68), class C proteins (30-80% bound to heat-shock granules, e.g. hsps 21, 17, 15) and class D proteins, which are minor heat-shock proteins only detected in structure-bound form. Major representatives are modified proteins, i.e. hsps 95, 80, 70 and 68 are phosphorylated and hsps 80, 74, 70 and 17 are methylated proteins (numbers 70, 80 etc. refer to 10(-3) Mr). Under heat-shock conditions synthesis of the proteins detected in control cells (25 degrees C proteins) exhibits two patterns. There are proteins with continued and proteins with discontinued synthesis. Synthesis of most of the latter proteins is resumed very rapidly after shift-down to 25 degrees C, even in the presence of actinomycin D. We conclude that reversible segregation of distinct mRNA species from the translation apparatus contributes to the heat-shock-specific pattern of protein synthesis in plants also.