XB-ART-55635Cell January 1, 2019; 176 (4): 805-815.e8.
Importin α Partitioning to the Plasma Membrane Regulates Intracellular Scaling.
Early embryogenesis is accompanied by reductive cell divisions requiring that subcellular structures adapt to a range of cell sizes. The interphase nucleus and mitotic spindle scale with cell size through both physical and biochemical mechanisms, but control systems that coordinately scale intracellular structures are unknown. We show that the nuclear transport receptor importin α is modified by palmitoylation, which targets it to the plasma membrane and modulates its binding to nuclear localization signal (NLS)-containing proteins that regulate nuclear and spindle size in Xenopus egg extracts. Reconstitution of importin α targeting to the outer boundary of extract droplets mimicking cell-like compartments recapitulated scaling relationships observed during embryogenesis, which were altered by inhibitors that shift levels of importin α palmitoylation. Modulation of importin α palmitoylation in human cells similarly affected nuclear and spindle size. These experiments identify importin α as a conserved surface area-to-volume sensor that scales intracellular structures to cell size.
PubMed ID: 30639102
PMC ID: PMC6368448
Article link: Cell
Genes referenced: ctnnb1 h2bc21 kif22 kif2a kifc1 kpna1 lmnb3 lypla1 lyplal1 porcn rpe tpx2
Antibodies: GFP Ab21 Histone H2B Ab10 Kif22 Ab1 Kif2a Ab1 Kifc1 Ab1 Lyplal1 Ab1 Porcn Ab1 Tpx2 Ab1
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|Figure 1 Palmitoylation Drives Importin α Plasma Membrane Association (A) Immunoblot of Xenopus eggs fractionated over a sucrose gradient and probed with importin α and beta-catenin antibodies. Importin α is found in the cytoplasm and also cofractionates with beta-catenin as a marker for plasma membrane mostly in the heavy membrane fraction. (B) Immunoblot of cytoplasm and membrane fractions isolated from stage 3 and stage 8 embryos and probed with importin α and Beta-catenin antibodies. A greater fraction of membrane-associated importin α is observed at stage 8. (C) Blot of immunoprecipitated recombinant wild-type and mutant importin α proteins (2S: S154A, S490A; NP: S154A, S490A C230A, C454A) retrieved from Xenopus egg extract and probed with importin α antibodies or streptavidin to quantify incorporation of biotin-labeled palmitate (Martin, 2013). (D) Blot of immunoprecipitated recombinant importin α retrieved from Xenopus egg extract following a 1 hour incubation with DMSO solvent, 10 μM palmostatin, or 1 μM Wnt-C59 and probed with importin α antibodies and streptavidin to quantify incorporation of biotin-labeled palmitate (Martin, 2013). (E) Fluorescence images of GFP-tagged wild-type or NP mutant importin α added to Xenopus egg extract, co-stained with FM4-64X to visualize plasma membrane lipid derived vesicles, in the presence of DMSO control or drugs that alter palmitoylation. Two vesicles are shown for each condition. GFP- importin α wt localization to vesicles was enhanced by palmostatin and inhibited by Wnt-C59, whereas GFP- importin α-NP did not co-localize with plasma membrane lipid vesicles under any condition. Scale bar, 10 μm.|
|Figure S1Importin α Fractionates with Plasma Membrane and Like Ras Is Palmitoylated, Related to Figure 1. (A) Left: Sucrose gradient of egg membranes to which recombinant GFP-importin α was added. Right: Fractions illuminated with blue light reveal that exogenous importin α co-fractionates at a much higher concentration with the plasma membrane-containing than the heavier membrane fraction (Hill et al., 2005). (B) Blot of immunoprecpitated Ras and importin α subjected to ABE chemistry to detect palmitoylation. Omission of 1 μM hydroxylamine (HA) serves as a negative control.|
|Figure 3 Importin α Palmitoylation Regulates Its Binding to NLS-Containing Cargos. (A) Fluorescence images and quantification of kif2a association with spindle microtubules in metaphase-arrested egg extract reactions containing 1 μM recombinant wild-type or NP mutant importin α in the presence of DMSO or 10 μM palmostatin. Importin α-NP reverts the increase in kif2a localization caused by importin α hyper-palmitoylation. Middle panel: Line scan quantification of fluorescence intensity across the length of 35 spindles normalized for length. Quantification shows mean ± SD from two extracts, ∗∗ = p < 0.0005. Right panel: Immunoblot of microtubules pelleted from metaphase-arrested egg extract reactions containing DMSO or 10 μM palmostatin probed with kif2a and tubulin antibodies. Hyperpalmitoylation of importin α enhances kif2a association with microtubules, mean ± SD from 2 experiments, p < 0.005. (B) Fluorescence images and quantification of nuclear lamin staining in interphase egg extract reactions containing 1 μM recombinant wild-type or NP mutant importin α in the presence of DMSO or 10 μM palmostatin. Importin α-NP reverts the decrease in nuclear accumulation caused by importin hyper-palmitoylation. Right panel: Quantification of the mean intensity of lamin B3 staining in 277 nuclei from 3 extracts, mean ± SD from two extracts, ∗∗ = p < 0.0005. Scale bars, 10 μm.|
|Figure S2 Effects of Palmostatin on Microtubule-Associated Proteins, Cargo Binding of Importin α and Its Partitioning of to the Droplet Surface, Related to Figures 3, 5, 6, and 7. (A) Line scan quantification of TPX2 immunofluorescence intensity with normalized spindle lengths upon addition of DMSO or 10 μM palmostatin. (B) Quantification of microtubule pelleting upon addition of DMSO or 10 μM palmostatin to spindle assembly reactions in egg extacts. Palmostatin increased the fraction of XCTK2, TPX2 and kif2a bound to microtubules, but did not affect chromokinesin Xkid. (C) Immunoprecipitation of kif2a and immunoblot of importin α following addition of DMSO or 10 μM palmostatin to egg extract. Palmostatin decreased the amount of importin α associated with kif2a. (D) Immunoprecipitation of GFP-lamin B3 and immunoblot of importin α following treatment of extracts with DMSO, 50 μM palmostatin or 10 μM Wnt-C59. Palmostatin decreased the amount of importin α associated with the nuclear lamin, while Wnt-C59 increased co-precipitating importin α. (E) Mean intensity ratio of importin α at the edge compared to the center in extract droplets encapsulated using synthetic or physiological lipids. Mean ± SD from 18 droplets, p < 0.005. (F) Mean intensity ratio of importin α at the cell membrane compared to the cell center in RPE-1 cells that have been treated with DMSO, palmostatin, or Wnt-C59. Mean ± SD from 30 cells, p < 0.05. ( G) Blot of HEK293 cells transfected with either control, LYPLA1 or PORCN siRNA.|