BAF-1 mobility is regulated by environmental stresses

Abstract

Barrier to autointegration factor (BAF) is an essential component of the nuclear lamina that binds lamins, LEM-domain proteins, histones, and DNA. Under normal conditions, BAF protein is highly mobile when assayed by fluorescence recovery after photobleaching and fluorescence loss in photobleaching. We report that Caenorhabditis elegans BAF-1 mobility is regulated by caloric restriction, food deprivation, and heat shock. This was not a general response of chromatin-associated proteins, as food deprivation did not affect the mobility of heterochromatin protein HPL-1 or HPL-2. Heat shock also increased the level of BAF-1 Ser-4 phosphorylation. By using missense mutations that affect BAF-1 binding to different partners we find that, overall, the ability of BAF-1 mutants to be immobilized by heat shock in intestinal cells correlated with normal or increased affinity for emerin in vitro. These results show BAF-1 localization and mobility at the nuclear lamina are regulated by stress and unexpectedly reveal BAF-1 immobilization as a specific response to caloric restriction in C. elegans intestinal cells.

FIGURE 1:

Eat-2 mutation and food deprivation reduce the mobile fraction of GFP::BAF-1 in L1 larvae. (A) FRAP analysis of GFP::BAF-1 in wild-type (blue) or in eat-2 (ad1116; red) C. elegans L1 larvae. n = 7 and 12, respectively. (B) FRAP analysis of GFP::BAF-1 in wild-type L1 (blue), wild-type L2 (red), or food-deprived (FD) L1 (green) animals. Error bars indicate SEM. x-axis: time; y-axis: relative fluorescence intensity (RFI). n = 15, 15, and 30 for L1, L2, and FD, respectively. Bleaching area: 2.8 μm².

FIGURE 2:

FLIP analysis of intestinal cells in L1 larvae reveals reduced GFP::BAF-1 mobility in response to dietary restriction, food deprivation, or 1-h heat shock. (A) FLIP analysis of GFP::BAF-1 in intestinal cells of L1 larvae in wild-type (top) or in eat-2 animals (bottom). Scale bar: 5 μm. (B) Mobility plot of the relative intensity of the GFP::BAF-1 in wild-type (green line) and eat-2 animals (purple). (C) Mobility plot of the relative intensity of the GFP::BAF-1 in C. elegans L1 larvae that were well fed (blue line), animals that were food deprived overnight (red line), or following 2-h recovery from FD (green line). FLIP analysis of emerin (D) HPL-1 (E) and HPL-2 (F) fused to GFP did not reveal any difference in mobility after food deprivation. (G) Mobility plot of the relative intensity of GFP::BAF-1 in wild-type animals with time following 1-h heat shock at 37°C. Error bars indicate SEM. For each experiment in (B), n = 7; in (C–G), n = 6. x-axis: time; y-axis: relative fluorescence intensity (RFI). The nuclei shown here were taken from the anterior part of the intestine. Bleaching area: 2 μm².

FIGURE 3:

Mass spectrometry analysis of GFP::BAF-1 Ser-4 phosphorylation before and after heat shock of asynchronous C. elegans populations. (A) BAF-1 peptides identified by mass spectrometry. The overall peptide coverage was 80 and 82% from control and heat shock samples, respectively. (B) Relative intensity of mass spectrometry spectra showing the Ser-4–phosphorylated and nonphosphorylated BAF-1 peptide CAAGPGSTGMSTSVK from control animals (top) and heat-shocked animals (bottom).

FIGURE 4:

In vivo analysis of wild-type and missense-mutant GFP::BAF-1 polypeptides. (A) Aligned amino acid sequences of BAF-1 (top) and human BAF (bottom), showing the missense mutations studied in this work (yellow arrow). (B) Fluorescence localization of GFP::BAF-1 (wild-type or mutant) protein in intestinal cell nuclei of L1 larvae. Scale bar: 5 μm. Each strain expresses a different GFP::BAF-1–based construct. The graph below each image shows the average intensity distribution across six nuclei (blue) on the same baseline as wild-type GFP::BAF-1 (red). (C and D) BIAcore analysis of the kinetics and equilibrium affinities of recombinant MBP::BAF-1, Ce-emerin residues 1–125, or Ce-lamin tail domain residues 388–566, each tested for binding to immobilized recombinant Ce-emerin residues 1–125 (C) or Ce-lamin R55H (D). χ² represents the mean square of the signal noise.

FIGURE 5:

BAF-1 mobility in L1 larvae is affected by combined heat shock and S4A-, S4E-, and K6A-mutated GFP::BAF-1. FRAP analysis of larvae L1 intestinal cells expressing GFP::BAF-1 wild-type and mutant GFP-BAF-1 before and after heat shock (1 h, 37°C). (A) Images of L1 larvae with no heat shock (control, top) or after 1 h heat shock (bottom). Results graphed in (B) as the mobile percentage before and after heat shock in L1 larvae intestinal cells. Error bars indicate SEM. Scale bar: 5 μm. n = 5–15 for each data point.

FIGURE 6:

GFP::BAF-1 immobilization is regulated by vrk-1. FLIP analysis of GFP::BAF-1 in animals grown on empty vector (EV) or vrk-1 RNAi (VRK) before and after heat shock (HS). Scale bar: 10 μm. Bleaching area: 3.1 μm².