Nuclear envelope irregularity is induced by RET/PTC during interphase

Abstract

Nuclear envelope (NE) irregularity is an important diagnostic feature of cancer, and its molecular basis is not understood. One possible cause is abnormal postmitotic NE re-assembly, such that a rounded contour is never achieved before the next mitosis. Alternatively, dynamic forces could deform the NE during interphase following an otherwise normal postmitotic NE re-assembly. To distinguish these possibilities, normal human thyroid epithelial cells were microinjected with the papillary thyroid carcinoma oncogene (RET/PTC1 short isoform, known to induce NE irregularity), an attenuated version of RET/PTC1 lacking the leucine zipper dimerization domain (RET/PTC1 Deltazip), H (V-12) RAS, and labeled dextran. Cells were fixed at 6 or 18 to 24 hours, stained for lamins and the products of microinjected plasmids, and scored blindly using previously defined criteria for NE irregularity. 6.5% of non-injected thyrocytes showed NE irregularity. Neither dextran nor RAS microinjections increased NE irregularity. In contrast, RET/PTC1 microinjection induced NE irregularity in 27% of cells at 6 hours and 37% of cells at 18 to 24 hours. RET/PTC1 Deltazip induced significantly less irregularity. Since irregularity develops quickly, and since no mitoses and only rare possible postmitotic cells were scored, postmitotic NE re-assembly does not appear necessary for RET/PTC signaling to induce an irregular NE contour.

Figure 1.

Lamin immunostaining pattern is unaltered in a cell microinjected 6 hours previously with cascade-blue-labeled dextran (cell marked with an arrow). Only lamin staining is shown. Conventional epifluorescence microscope, final magnification ×1000.

Figure 2.

Lack of cell recovery after microinjection can be mostly accounted for by an early cell death, associated with a characteristic shrunken appearance by phase contrast microscopy. Corresponding phase contrast (A) and superimposed fluorescein-dextran (green) and ethidium bromide (red) (B) fluorescent images show that microinjected cells that remain viable have a grossly normal morphology, whereas cells that die as a result of microinjection (red) show a characteristic flattened morphology and lose much of the microinjected fluorescent dextran. Final magnification, ×50.

Figure 3.

Dead cells tend to detach during immunostaining, while cells expressing microinjected plasmids are viable and do not detach. Phase contrast (A) and superimposed phase contrast and ethidium bromide (B) shows three dead cells in a living culture of normal thyroid culture 12 hours after microinjection of RET/PTC1. Approximately the same field is shown following fixation and immunostaining for RET (C). The three dead cells had detached, and the RET/PTC1-expressing cells (including a binucleated cell) were viable at the time of fixation. Final magnification, ×150.

Figure 4.

A rare cell with a fragmented nuclear envelopes (upper right) is encountered that likely died early as a result of the microinjection procedure itself. The appearance is distinctly different from the appearance of viable cells. Only lamin staining is shown. Conventional epifluorescence microscope, final magnification, ×1000.

Figure 5.

Microinjection of RET/PTC Δzip (A to C) causes no change in nuclear contour in three cells, whereas microinjection of RET/PTC1 (D to F) induces nuclear envelope irregularity within 18 hours. Anti-RET antibody (red) in A, D, and the inset to F shows unambiguous staining of microinjected cells; B, C, E, and F show the lamin immunostaining of the corresponding fields. The appearance of the three nuclei that had been microinjected with RET/PTC Δzip 6 hours previously (arrows in B) is not different from the surrounding non-injected cells. C: Higher magnification of the upper two cells of B. In contrast, one cell that had been microinjected with RET/PTC1 18 hours previously (D to F) shows an irregular nucleus in the expressing cell. This cell can be seen in E (arrow) to be at a higher plane than the surrounding non-injected cells. F shows the same microinjected cell at higher magnification in focus by conventional epifluorescence and the inset shows the cell by confocal microscopy. Magnifications: A and B, ×300; D and E, ×400; C and F, ×1000.

Figure 6.

Nuclear envelope irregularity is induced by RET/PTC1 within 6 hours of microinjection. The proportion of cells (shown above each of the histograms) with NE irregularity at 6 and 18 to 24 hours after microinjection of RET/PTC1 is significantly higher (P < 0.001) than non-injected cells, dextran-injected cells, or RAS-injected cells. 18 hours after the microinjection of RET/PTC Δzip, significantly fewer cells (P < 0.01) have nuclear envelope irregularity than cells microinjected 18 hours previously with RET/PTC1, but significantly more than non-injected, dextran-injected, or RAS-injected cells. Six hours after RET/PTC Δzip microinjection, the proportion of cells with NE irregularity is statistically (P < 0.05) less than at 6 hours after RET/PTC1 microinjection. Irregularity after RAS microinjection is not significantly (NS) different from controls.

Figure 7.

No evidence for an intervening mitosis can be seen in two cells with irregular nuclei 6 hours after microinjection of RET/PTC1. The two cells are separated from each other by two other non-injected cells. A: Anti-RET immunostain. B: Lamin immunostaining of the same field. C: Confocal micrograph of the same cell marked with an arrow in B. The confocal micrograph shows superimposed RET immunostain product (red) and lamin (green) staining and it shows with better resolution the NE irregularity consisting of numerous longitudinal folds. A and B: Conventional epifluorescence micrographs at ×400 final magnification. C: Confocal image at ×1000 final magnification.

Figure 8.

Nuclear envelope irregularity occurs in non-mitotic cells that appear singly, rather than in postmitotic doublets. To try to eliminate scoring of a cell that could have passed through mitosis, a subset of the results shown in Figure 1 was scored as to whether an expressing cell was next to another expressing cell. NE irregularity 18 hours after RET/PTC1 microinjection in single cells is statistically higher than RAS or RET/PTC Δzip at either time point, and at 6 hours after RET/PTC1 microinjection, the rate of single cells with irregular NE contours is significantly higher than RAS at the 18-hour time-point.