Alterations in nuclear matrix ultrastructure of G1 mammalian cells following heat shock: Resinless section electron microscopy, biochemical, and immunofluorescence studies

Abstract

Heat shock is known to inhibit vital nuclear functions associated with DNA and RNA metabolism. It has been proposed that the reported heat-induced excess protein accumulation in the nuclear matrix (NM) fraction may alter NM sites crucial for DNA and RNA processing. To test this hypothesis, we examined the fine structure of the NM in synchronous populations of G1 Chinese hamster ovary cells before and after heating by using the technique of resinless section electron microscopy. Heat did induce morphological alterations in the NM. The NM of control cells contained a honeycomb-like arrangement of fibers after chromatin removal. Following heat shock, NMs appeared as more highly anastomosing networks of polymorphic fibers and an overall increase in electron density was observed. Residual nucleoli from heated NMs underwent alterations in distributions of electron density both internally and at their peripheries. The increase in electron density observed in heated NMs was accompanied by an increase in protein mass and a relatively smaller increase in RNA mass as indicated by parallel sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and isotopic labeling (protein/DNA and RNA) studies. Some excess protein accumulation could also be directly localized onto NM fibers by use of antibodies to heterogeneous ribonucleoprotein complex antigens. It is concluded that alterations of NM fine structure can reflect the heat-stressed state of the cell, may account for the heat-induced inhibition of nucleic acid metabolism, and may be useful as an indicator of physiological or pathological stress in general.

Recovery of nuclear matrix ultrastructure of interphase CHO cells after heat shock

“…The ultrastructure of these matrices is similar to that previously seen in CHO nuclear matrices that were processed and sectioned in suspension (Wachsberger and Coss, 1993). In our previous study, an analysis of DNA, RNA, and protein content revealed that 1-3% of cell DNA, 13-16% of cell RNA, and &12% of cell protein remained associated with the matrix preparations.…”

“…In this range, alterations in NM ultrastructure were clearly observed immediately after heating. As previously reported, these alterations included 1) altered conformation from a loosely woven fiber network to a highly anastomosing network, 2) altered conformation of nucleolar bodies with a concomitant increase in electron density, and 3) an overall increase in NM electron density (Wachsberger and Coss, 1993). Figure 2 shows the representative morphologies of NM obtained from GI CHO cells 30 min at 37°C after a heat dose at 45°C for 20 min (Fig 3a) and 20 hr follow-ing the 45"C, 20 min heat dose (Fig.…”

“…It has been proposed that this excess nuclear protein may play a central role in heat-induced ceIl death by altering chromatin organization on the nuclear matrix and thereby DNA-associated processes (Roti Roti and Laszlo, 1988;Laszlo, 1992). We have demonstrated that hyperthermia induces ultrastructural changes in nuclear matrix architecture that coincide with heat-induced increases in nuclear matrix protein (Wachsberger and Coss, 1993). Changes in NM architecture immediately following heating included 1) altered conformation from a loosely woven fiber network to a highly anastomosing network, 2) altered conformation of nucleolar bodies with a concomitant increase in electron density, and 3) an overall increase in NM electron density.…”

Lamin B is a prompt heat shock protein

“…Recent evidence suggests that the nuclear matrix represents a critical target for heat cytotoxicity and that nuclear matrix and nuclear matrix-associated proteins figure prominently in the response to hyperthermia (Warters et al, 1986;Laszlo et al, 1992;Roti Roti and Turkel, 1994;VanderWaal et al, 1996;Roti Roti et al, 1997). Hyperthermia causes changes in nuclear matrix architecture (Wachsberger and Coss, 1993), and these structural changes, as well as changes in the abundance and conformation of specific nuclear matrix proteins, could be critical for the continuation or resumption of nuclear metabolism after heat shock (Henle and Leeper, 1979;Wong and Dewey, 1982). For a better understanding of the role of lamin B in the heat response, careful studies are necessary to determine the rate and extent of incorporation of newly synthesized lamin B into the nuclear lamina during and after heat shock, the rate of recovery of lamin B content to normal levels, and the effects of dysregulation of lamin B synthesis on the recovery of DNA and RNA metabolism.…”

“…Structural and functional alterations to the nuclear matrix have been hypothesized to play a central role in heat-induced cell killing (Roti Roti and Laszlo, 1988;Roti Roti et al, 1997). Recent evidence suggests that protein accumulation induced by heat shock could alter DNA and RNA metabolism at specific nuclear matrix sites (Wachsberger and Coss, 1993).…”