Concurrent collapse of keratin filaments, aggregation of organelles, and inhibition of protein synthesis during the heat shock response in mammary epithelial cells.

The sequence of heat shock-induced perturbations in protein synthesis and cytoskeletal organization was investigated in primary cultures of mouse mammary epithelial cells (MMEC). Exposure of the cells to 45 degrees C for 15 min caused a marked inhibition of protein synthesis through 2 h after heart. Resumption of protein synthesis began by 4 h, was complete by 8 h, and was accompanied by induction of four major heat shock proteins (HSPs) of 68, 70, 89, and 110 kD. Fluorescent cytochemistry studies indicated that heat shock elicited a reversible change in the organization of keratin filaments (KFs) and actin filaments but had a negligible effect on microtubules. Changes in the organization of KFs progressed gradually with maximal retraction and collapse into the perinuclear zone occurring at 1-2 h after heat followed by restoration to the fully extended state at 8 h. In contrast, actin filaments disappeared immediately after heat treatment and then rapidly returned within 30-60 min to their original appearance. The translocation of many organelles first into and then away from the juxtanuclear area along with the disruption and reformation of polyribosomes were concurrent with the sequential changes in distribution of KFs. The recovery of the arrangement of KFs coincided with but was independent of the resumption of protein synthesis and induction of HSPs. Thermotolerance could be induced in protein synthesis and KFs, but not in actin filaments, by a conditioning heat treatment. Neither protein synthesis nor induction of HSPs was necessary for the acquisition of thermotolerance in the KFs. The results are compatible with the possibility that protein synthesis may depend on the integrity of the KF network in MMEC. Heat shock thus can efficiently disarrange the KF system in a large population of epithelial cells, thereby facilitating studies on the functions of this cytoskeletal component.

Effect of growth state and heat shock on nucleolar localization of the 110,000-Da heat shock protein in mouse embryo fibroblasts.

We have shown previously that the mammalian 110,000-Da heat shock protein (hsp110) associates with nucleoli in several cell types and that in 2-day postconfluent mouse 10T1/2 cells, a segregation of the antigen from the nucleolar phase-dense body is seen (J.R. Subjeck, T. Shyy, J. Shen, and R.J. Johnson. J. Cell Biol. 97: 1389-1395, 1983). Here we further characterize the nucleolar segmentation of hsp110 in mouse 10T1/2 and 3T3 cells with respect to the formation of this structure in dense cultures and investigate the behavior of this protein following conditions (serum deprivation, actinomycin D, and heat shock) known to affect the functional and morphological integrity of the nucleolus. It is shown that in addition to its nucleolar locale, an affinity of hsp110 for the nonnucleolar, nuclear compartment in actively proliferating cells is also observed. When proliferating cells are treated with actinomycin D (1 microgram/ml) for 8 h, hsp110 separates from the nucleolar phase-dense body to form a fluorescent nucleolar cap which resembles that seen in confluent cultures. This drug also results in a disappearance of hsp110 from the nucleoplasm. Incubation of cells for 24 h in media without serum also results in the nucleolar segmentation of hsp110 and a reduction in nucleoplasmic staining. A moderate nonlethal heat treatment does not lead to segmentation of hsp110 in proliferating cells but conversely results in a transient reversal of segmentation in confluent cultures. Examination of segmented nucleoli of postconfluent cells by immunoelectron microscopy reveals that hsp110 is associated with the fibrillar component of these nucleoli, the site of ribosomal DNA.

Recovery of vasomotor response in human spinal cord transection.

Vasomotor responses of the skin of the thumb and the big toe were measured in normal subjects and patients with spinal transection at a neutral ambient temperature of 22.0 +/- 0.5 degrees C, and in a cool (12.0 +/- 0.5 degrees C) and warm (32.0 +/- 0.5 degrees C) environment. The vasomotor response of the hand and foot to the cooling and warming of part of the opposite upper or lower extremity was also recorded. Spinal transection at T5-11 abolishes acutely all vasomotor responses in the paraplegic lower extremities, but does not alter the responses in the upper extremities. By 4 months, the vasomotor tone in the lower extremities at a neutral ambient temperature returned to normal values as did the response to a cool and warm environment. The crossed vasomotor reflex to cooling and warming one lower extremity recovers more slowly, requiring a period of 18 months for complete recovery. The slower recovery of the vasomotor reflex in spinally transected man than in similarly treated dogs is though to be due to the greater spinal shock in the former. The recovery of vasomotor responses in the paraplegic limbs to cooling and warming after thoracic transections suggests that these responses are primitive and powerful thermoregulatory mechanisms.

Stress protein systems of mammalian cells.

Living organisms are known to react to a heat stress by the selective induction in the synthesis of several polypeptides. In this review we list the major stress proteins of mammalian cells that are induced by heat shock and other environments and categorize these proteins into specific subgroups: the major heat shock proteins, the glucose-regulated proteins, and the low-molecular-weight heat shock proteins. Characteristics of the localization and expression of proteins in each of these subgroups are presented. Specifically, the nuclear/nucleolar locale of certain of the major heat shock proteins is considered with respect to their association with RNA and the recovery of cells after a heat exposure. The induction of these major heat shock proteins and the repression of the glucose-regulated proteins as a result of reoxygenation of anoxic cells or by the addition of glucose to glucose-deprived cultures is described. Changes in the expression of these protein systems during embryogenesis and differentiation in mammalian and nonmammalian systems is summarized, and the protective role that some of these proteins appear to play in protecting the animal against the lethal effects of a severe heat treatment and against teratogenesis is critically examined.

Analysis of the expression of the two major proteins of the 70 kilodalton mammalian heat shock family.

This study compares the expression after heat shock of the two major variants of the mammalian 70 kilodalton heat shock family in three separate systems. The ability of wild type and temperature sensitive mutant (ts85) FM3A cells to elicit a heat shock response following a 45 degrees C, 12 min exposure was examined. The ts85 cells were found to be both significantly more thermosensitive than parent FM3A cells and to induce a 66kDa heat shock protein (hsp66) not visibly synthesized in the parent line by this exposure. However, a constitutive (synthesized at 37 degrees C) 68kDa heat shock protein (hsp68) is comparably induced in both cell lines after heat. A relationship between the severity of the heat exposure as seen by the cell and hsp66 expression is suggested and tested in Chinese hamster ovary cells. In CHO cells a brief 45 degrees C heat shock induces the constitutive hsp68 (but not hsp66), while longer and more severe exposures are required for the expression of hsp66. The induction of these two proteins is also examined in situ in mouse skeletal muscle. In this case both hsp66 and hsp68 are induced following comparatively mild heat treatments, and the 'threshold' for hsp66 induction observed in cultured cells either does not occur or is greatly reduced. However, once again, hsp68 is naturally synthesized at 37 degrees C while hsp66 appears to be de novo synthesized after heat shock.