Dynamics of lamin-A processing following precursor accumulation

Abstract

Lamin A (LaA) is a component of the nuclear lamina, an intermediate filament meshwork that underlies the inner nuclear membrane (INM) of the nuclear envelope (NE). Newly synthesized prelamin A (PreA) undergoes extensive processing involving C-terminal farnesylation followed by proteolysis yielding non-farnesylated mature lamin A. Different inhibitors of these processing events are currently used therapeutically. Hutchinson-Gilford Progeria Syndrome (HGPS) is most commonly caused by mutations leading to an accumulation of a farnesylated LaA isoform, prompting a clinical trial using farnesyltransferase inhibitors (FTI) to reduce this modification. At therapeutic levels, HIV protease inhibitors (PI) can unexpectedly inhibit the final processing step in PreA maturation. We have examined the dynamics of LaA processing and associated cellular effects during PI or FTI treatment and following inhibitor washout. While PI reversibility was rapid, with respect to both LaA maturation and associated cellular phenotype, recovery from FTI treatment was more gradual. FTI reversibility is influenced by both cell type and rate of proliferation. These results suggest a less static lamin network than has previously been observed.

Figure 1. HIV PIs block LaA maturation, which is reversed rapidly following PI washout.

(A) Blotted with PreA specific and LaA/C antibodies, extracts from Saos-2 cells treated with vehicle (DMSO), Tip, Nel, Ata, Ind and Lop reveal different levels of PreA accumulation. Percentage of mature LaA is listed under each lane. (B) Immunoblot of extracts from Saos-2 cells treated with Lop followed by washout indicates that half time of LaA maturation is approximately 3 hrs. The processing rate of the overnight ³⁵S Cys/Met labeled LaA following Lop washout is similar. Percentage of mature LaA is listed under each lane. (C) Immunofluorescence microscopy of Saos-2 cells double labeled with the antibodies again PreA and Nup153. PreA is accumulated on the NE after Lop treatment and disappears following Lop washout. All images are taken at the same exposure time. In the merged images, DNA, revealed by staining with Hoechst dye, is shown in blue. Bar, 15µm.

Figure 2. Recovery from aberrant cellular phenotypes is delayed following Lop washout.

(A) Immunofluorescence microscopy of Saos-2 cells. Altered interphase nuclear morphology and abnormal accumulation of LaA/C and emerin in the cytoplasm are evident after Lop treatment and recover within 15 hrs following Lop washout. (B) The aberrant cytoplasmic aggregates after Lop treatment contain LaB1, predominantly colocalized with LaA/C. (C) Nuclear circularity following Lop washout is significantly altered by PI treatment and following washout either without (grey bars) or with cycloheximide (white bars) (error bars = SEM; N = >96). (D) Double labeled with antibodies against LaA/C and LaB1, Saos-2 cells exhibit normal nuclear morphology after Lop treatment following LaA/C RNAi. (E) Nuclear circularity is higher in LaA/C RNAi treated cells (error bars = SEM; N = >65). (F) Nuclear components such as LaA/C, emerin and LaB1 are diffuse in control cells during metaphase. (G) Lop treatment leads to aberrant aggregation of LaA/C adjacent to metaphase chromosomes. Emerin and LaB1 are also retained in these aggregates (white arrows). (H) Measurements of percentage of cells with metaphase aggregates indicate that mitotic abnormality recovers by ∼15 hrs after Lop washout (error bars = SEM; N = 3, >75 cells counted each experiment). (I) LaA/C, emerin and LaB1 localize on the NE in early G1. (J) Lop treatment leads to aberrant aggregation of LaA/C in the cytoplasm in early G1. These aggregates also contain emerin and LaB1 (white arrows). (K) Measurements of percentage of cells with early G1 aggregates indicate that recovery of cytoplasmic abnormality is ∼15 hrs after Lop washout (error bars = SEM; N = 3, >75 cells counted each experiment). Bars, 5µm.

Figure 3. Recovery of nuclear morphology following Lop washout is time dependent and enhanced in cycling cells.

(A) Probed with LaA/C antibody, immunoblot of Saos-2 cells treated with increased concentrations of Lop from 0 to 40 µM reveals increased PreA accumulation in parallel. Percentage of mature LaA is listed under each lane. (B) Measurements of interphase nuclear circularity (error bars = SEM; N = >80), percentage of cells with metaphase and early G1 aggregates (error bars = SEM; N = 3, >75 cells counted each experiment) reveal that the effects of 5µM Lop differs significantly from 20µM Lop, with similar level to vehicle (DMSO) control. (C) Accumulated PreA in Lop treated cells complete its maturation by 7 hrs following drug washout. Mitomycin C treatment prior to Lop removal does not affect PreA processing rate. Percentage of mature LaA is listed under each lane. (D) Measurement of nuclear circularity indicates that mitomycin delays recovery of aberrant nuclear shape at 15 hrs following Lop washout (error bars = SEM; N = >92).

Figure 4. Nonfarnesylated-PreA accumulated at the NE by prenylation inhibitors gradually matures following drug washout.

(A) Immunofluorescence microscopy of Saos-2 cells reveals significant accumulation of nonfarnesylated-PreA on the NE after a treatment of Lov or FTI-277. DNA, revealed by staining with Hoechst dye, is shown at bottom column. Bar, 5µm (B) Immunoblot of extracts from Saos-2 cells treated with Lov followed by washout indicates that half-time of PreA maturation is ∼7 hrs. Cyclohexamide chase upon release from Lov block enhances maturation. Accumulations of slower migrating non-farnesylated HDJ-2 regain farnesylation by 2 hrs following Lov washout. Processing rate of overnight ³⁵S Cys/Met labeled LaA following Lov removal is similar to unlabeled total population. (C) In similar experiments with FTI-277, the half-time of PreA maturation following FTI-277 washout is ∼15 hrs in total lysates, ∼7 hrs with cyclohexamide chase and ∼12 hrs in an overnight ³⁵S Cys/Met labeled population. HDJ-2 is farnesylated at a faster rate than LaA is processed upon FTI-277 washout. (D) Immunoblot of extracts of Saos-2 cells probed with LaA antibody. In contrast to the incomplete PreA accumulation by FTI-277 treatment, a combinatorial treatment of FTI-277 and GGTI-2147 or Lov-alone lead to more complete accumulation of non-prenylated PreA. Percentage of mature LaA or HDJ-2 is listed under each lane.

Figure 5. Aberrant cellular phenotypes recover rapidly in PI treated cells following washout of FTI and GGTI.

(A) Labeled with LaA/C antibody, immunofluorescence microscopy of Saos-2 cells reveals that the combination of FTI-277 and GGTI-2147, but not FTI-alone, rectify aberrant nuclear morphology and abnormal nucleoplasmic aggregation caused by Lop. Release of FTI and GGTI enhance this phenotypic rebound. Bar 10µm. (B) Measurements of nuclear circularity and percentage of cells with metaphase and early G1 aggregates reveal that corresponding cellular phenotypes are reversed by 15 hrs following FTI-277 and GGTI-2147 washout and by 24 hrs following FTI-277 washout (error bars = SEM; N = 3, >75 cells counted each experiment).

Figure 6. LaA and progerin are slow to mature in HGPS cells following FTI washout.

(A) FTI-277 treatment of HGPS cells for 96 hrs inhibits abnormal nuclear morphology as observed by LaA/C imunofluorescence (error bars = SEM; N = >80). Bar, 10µm. (B) After a 9 day FTI-277 treatment, ∼50% of the LaA is PreA and nonfarnesylated-progerin (non-farΔ50). In contrast FTI-277 and GGTI-2147 lead to a more complete accumulation of PreA and non-farΔ50. Following FTI or FTI and GGTI washout, mature LaA (matA) and farnesylated progerin (farΔ50) slowly recover, but at relatively similar rates. Percentage of mature LaA or LaAΔ50 is listed above or below each lane, respectively.