Abstract
Non-centrosomal microtubule-organizing centres (ncMTOCs) have a variety of roles that are presumed to serve the diverse functions of the range of cell types in which they are found. ncMTOCs are diverse in their composition, subcellular localization and function. Here we report a perinuclear MTOC in Drosophila fat body cells that is anchored by the Nesprin homologue Msp300 at the cytoplasmic surface of the nucleus. Msp300 recruits the microtubule minus-end protein Patronin, a calmodulin-regulated spectrin-associated protein (CAMSAP) homologue, which functions redundantly with Ninein to further recruit the microtubule polymerase Msps—a member of the XMAP215 family—to assemble non-centrosomal microtubules and does so independently of the widespread microtubule nucleation factor γ-Tubulin. Functionally, the fat body ncMTOC and the radial microtubule arrays that it organizes are essential for nuclear positioning and for secretion of basement membrane components via retrograde dynein-dependent endosomal trafficking that restricts plasma membrane growth. Together, this study identifies a perinuclear ncMTOC with unique architecture that regulates microtubules, serving vital functions.
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Data availability
The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files. Data not included are available from the corresponding authors upon reasonable request.
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Acknowledgements
We thank Megraw laboratory members for numerous discussions and critiques of the manuscript; M. Rolls, W.-M. Deng, J. Raff, R. Vale, T. Volk, N. Sherwood, R. Basto, N. Rusan, E. Lécuyer, T. Avidor-Reiss, M. Ringuette, D. Lerit, S. Rogers, J. Wakefield, H. Nakanishi and V. Gelfand for antibodies and Drosophila stocks; and Bloomington Drosophila Stock Center, Vienna Drosophila Resource Center, Kyoto Stock Center for Drosophila stocks and the Developmental Studies Hybridoma Bank, University of Iowa, for antibodies. We are grateful for funding from NIH grants R15GM119078 and R15HD099648 (T.L.M).
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T.L.M. and Y.Z. designed the study. Y.Z. performed and analysed most experiments. R.A.B. and J.V.C. performed and analysed centrosomal protein staining. C.Z. performed and analysed coimmunoprecipitation assays. E.M.W. imaged and analysed transmission electron microscopy data. Y.Z. and T.L.M wrote and revised the manuscript with constructive input from all authors.
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Extended data
Extended Data Fig. 1 Fat body MTs are stabilized.
(a) Additional EM images, related to Fig. 1c. (b) Images of IF stained fat body cells with or without cold treatment for 1 hr, which did not cause significant reduction of the MT array. (c, d) IF staining shows that the fat body contains MTs that are acetylated (c) and polyglutamylated (d), two post-translational modifications attributed to stabilized MTs. Genotype details are in Supplementary Table 3. Experiments were performed once in (a), twice in (b–d) with similar results. Scale bar, 1 µm (a), 20 µm (b–d).
Extended Data Fig. 2 Survey of localization of centrosomal proteins and MT regulators at the fat body ncMTOC.
Images of fat body cells stained for Cnn and counterstained in green for the indicated proteins using either antibody staining or expression from a fluorescent protein-tagged transgene as indicated. The insets are Cnn staining as positive control. See Supplementary Table 1 for the complete list of proteins assayed and the summary of their localization at the MTOC as well as the information about promoters used to drive transgenes. Anti-GFP or anti-RFP staining was applied for GFP-tagged or RFP-tagged transgenes. The centriolar proteins are core centriolar proteins. The “PCM + centriolar” proteins are proteins that reside in both compartments or straddle both compartments and are known to function in PCM organization. The “MT regulatory” are effector proteins with established roles in regulating MT assembly or anchoring. Since some of the transgenic proteins were expressed ectopically, it is possible that some of the positive localizations determined in this manner do not reflect endogenous protein localization. Collectively, the fat body MTOC contains some proteins in common with the centrosome PCM, but also some distinct components including Patronin. Note that Ana1-GFP image in Fig. 3b is reproduced from this figure. Genotype details are in Supplementary Table 3. Staining experiments were performed twice with similar results. Scale bar, 20 µm.
Extended Data Fig. 3 Single or double mutant combinations of centrosomal protein genes do not overtly impair MT assembly at the fat body ncMTOC.
Fat body cells were stained for DNA (Hoechst) and filamentous actin (CF568-Phalloidin) to assay for nuclear centricity (top panels). MTs (anti-α-tubulin) and DNA (DAPI) were stained to assay MT organization with respect to nuclei (bottom panels). None of the single or double mutants tested shows defects in nuclear positioning or MT assembly at the MTOC. Nuclear positioning (top panel) are quantified in Fig. 3a. Genotype details are in Supplementary Table 3. Experiments were performed twice with similar results. Scale bar, 50 µm (top panels), 20 µm (bottom panels).
Extended Data Fig. 4 γ-tubulin is not required for MT assembly at the fat body ncMTOC.
(a) Western blot detection of γ-tubulin in wild-type and γTub23C mutant larval fat body and brain lysates. Wild type is w1118, γTub mutant is γ-Tub23CA15-2/Df (2 L)JS17. Triose-phosphate isomerase (TPI) is the loading control. γ-tubulin levels, as measured from the shown blot, were about 120-fold lower in wild-type larval fat bodies compared to brains when normalized against Tpi. (b) Western blot detection of γ-tubulin in whole larval lysates from wild-type and three independent γTub23C RNAi lines driven by two ubiquitous strong promoters: Tub-Gal4 and with Act5c-Gal4 plus UAS-Dicer-2. (c) Images showing γ-tubulin staining in γ-Tub23CGL01171 RNAi fat body clone marked by His-RFP. (d, d’) Images showing γ-tubulin staining in wild-type (d) and γ-Tub23C mutant (d’) fat body at different developmental stages as indicated. (e, e’) Images showing nuclear positioning and MT assembly in wild-type (e) and γ-Tub23C mutant (e’) fat body cells. (f) Images showing MT regrowth in wild-type and γ-Tub23C depleted fat body cells from γ-Tub23CGL01171 RNAi clones marked by His-RFP. Control: no vinblastine treatment; the other three groups were treated with vinblastine and underwent MT regrowth for 0, 5, 30 min as indicated. The western blots in a and b, staining in c, d, d’, e, e’, f were repeated twice with similar results. Full blots for a, b are shown in Source Data Extended Data Fig. 4_Uncropped Western Blots. Scale bar, 50 µm (left panels in c, top panels in d, e’, top panels in f), 10 µm (right panels in c, bottom panels in d, e’), 20 µm (bottom panels in f).
Extended Data Fig. 5 Msps localization at the fat body ncMTOC does not require TACC, AurA or Ncd.
(a) Staining for Msps in msps RNAi fat body clones marked by His-RFP. Arrow indicates the loss of Msps in an msps RNAi cell. (b) Msps localization at centrosomes or spindle poles relies on TACC, Aurora A kinase phosphorylation of TACC, and the Kinesin-14 motor Ncd. None of these components are required for Msps localization at the fat body ncMTOC (top and middle panels), or for fat body nuclear positioning (bottom panels). Fat bodies were stained with antibodies against Msps and Cnn, or stained for DNA (DAPI), MTs using a FITC-conjugated DM1A antibody, and actin with CF568-conjugated phalloidin. Staining was repeated twice in a, three times in b with similar results. Nuclear positioning in b was quantified in Fig. 3a. Genotype details provided in Supplementary Table 3. Scale bar, 50 µm (a, bottom panels in b), 10 µm (top panels in b).
Extended Data Fig. 6 Patronin cooperates with Nin but not with γ-tubulin or other known Patronin interactors in fat body.
(a) Western blot detection of Patronin in larval fat body lysates from wild-type and Patronin knockdown. Arrows indicate the established Patronin isoform at 180 kDa, and another putative isoform at approximately 65 kDa. (b) MT staining in wild-type, single knockdown of Patronin or Klp10A (Kinesin-13), and double knockdown of Patronin plus Klp10A. (c) Mutants for MT severing enzymes katanin p60 or katanin p60-like 1 do not affect nuclear positioning (top) or MT assembly (bottom). (d) Knockdown of MT severing enzymes kat80 (c) or spastin (d) or double knockdown of kat80 plus spastin does not affect nuclear positioning (top) or MT assembly (bottom). (e) Mutant for dgt4 does not affect nuclear positioning (left) or MT assembly (right). (f) γTub23C mutation together with Patronin RNAi does not significantly affect nuclear positioning (left) or radial MTs (right). (g) Western blot detection of Nin in larval brain lysates from wild-type, Nin RNAi, and Nin1 mutant. (h) Nin staining in Nin RNAi fat body clone marked by His-RFP. (i) Nin, Patronin and MTs in γTub23C mutant fat body. (j) Double knockdown of Nin and γTub23C, or Nin and gcp3 does not affect nuclear positioning and MT assembly in fat body clones. The western blots in a and g were performed twice with similar results. Full blots are shown in Source Data Extended Data Fig. 6_Uncropped Western Blots. The staining were repeated three times in (b, c, e, i), twice in (d, f, h, j) with similar results. Scale bar, 20 µm (b, bottom panel in c, middle and bottom panels in d, right panel in h, i, top panels in j), 50 µm (top panels in c–e, left panels in f, h), 10 µm (magnified panels in j).
Extended Data Fig. 7 Shot depletion generates an ectopic MTOC and results in severe and pleiotropic disruption of subcellular compartments.
(a) shot knockdown results in a reorganization of the MTOC from the nuclear surface to a centrosome-like focus in the cytoplasm (arrows). (b, c, d) Cnn (b), γTub23C (c) and Klar (d) are not delocalized from the nuclear surface to the ectopic MTOC (arrows) generated by shot knockdown. Arrow in c indicates no γTub23C-GFP enrichment in the ectopic centrosome-like MTOC. (e) Actin ‘collapses’ into an aggregate (arrows) near the MTOC after shot knockdown. Note that nuclear centricity is also affected. Inset shows the merged image with MTs. (f, g) Nuclear morphology (f) and organization of organelles (g) are disrupted in shot knockdown fat bodies. Arrows point to accumulation of ER (RFP-KDEL), Golgi (ManII-GFP) and mitochondria (anti-ATP5α) near actin aggregates. (h) shot knockdown results in accumulation of basement membrane components in the cytoplasm, proximal to the ectopic MTOC (arrows). (i) Deposition of collagen IV α2 (Vkg-GFP, arrows) on the wing disc is decreased when shot is knocked down in fat body cells. (i’) Quantitation of Vkg-GFP in (i). Data are the means ± s.e.m (n = 6 independent experiments), p = 0.000088 (****) by two-tailed Student’s t-test. Statistical details are shown in Source Data Extended Data Fig. 7_Statistical Source Data. Experiments in a–h were performed three times with similar results. Genotype details are in Supplementary Table 3. Scale bar, 50 µm (a, e–i), 20 µm (b–d).
Extended Data Fig. 8 Supporting data for secretion of basement membrane components and retrograde trafficking as well as plasma membrane growth.
(a) Images showing fat body accumulation of different basement membrane components after disruption of MTs in fat body cells with β-Tub56D RNAi or spastin overexpression. (b) Images showing no accumulation of different basement membrane components after actin disruption in fat body by Arp2 RNAi. (c) Images showing that GFP-Rab5 endosomes shift from their perinuclear location (yellow arrowheads) to the plasma membrane site (white arrows) after inactivation of dynein activity (Dynamitin overexpression). Conversely, dynamin loss (shi RNAi) disrupts endocytic vesicle budding, causing a loss of GFP-Rab5 vesicles at the plasma membrane. (d) Images showing fat body accumulation of Vkg-GFP at the plasma membrane after inactivation of dynein activity either by Dhc64c RNAi or Dynamitin overexpression, but not after inactivation of kinesin-1 activity by Kinesin heavy chain (Khc) or Kinesin light chain (Klc) RNAi. Experiments in a–d were repeated twice with similar results. Genotype details are in Supplementary Table 3. Scale bar, 50 µm.
Extended Data Fig. 9 Summary and model for perinuclear non-centrosomal MTOC assembly and function in fat body cells.
(a) Drosophila fat body cells, a differentiated cell type analogous to human adipocytes and liver, assemble a perinuclear ncMTOC that organizes dense circumferential MTs and radial MTs. The radial MTs are polarized, emanating from the nuclear surface (minus-end) towards the plasma membrane (plus end). N: nucleus. (b) The perinuclear ncMTOC has unique architecture and MT assembly mechanisms. Msp300/Nesprin anchors the ncMTOC at nuclear surface, requiring and recruiting Shot, and is epistatic to Patronin/CAMSAP. Patronin and Nin cooperate to recruit Msps for radial MT elongation independently of γ-tubulin. Domains are shown for each protein. (c) The perinuclear ncMTOC has two critical physiological functions: nuclear positioning and control of plasma membrane growth via retrograde trafficking. The fat body perinuclear ncMTOC controls endosomal retrograde trafficking in coordination with minus-end directed dynein to restrict plasma membrane overgrowth. Disruption of the ncMTOC or inactivation of dynein blocks retrograde membrane trafficking, leading to excessive plasma membrane growth and convoluted “thickened” plasma membranes. Consequentially, secreted BM components are trapped within the convoluted plasma membrane folds. The entrapment of BM proteins in fat body cells leads to reduced BM deposition in destination tissues, including imaginal discs and brains (not depicted).
Supplementary information
Supplementary Tables
Supplementary Tables 1 through 5. 1. Survey of fat body ncMTOC components and key assembly regulators. 2. Relevant genotypes of strains and stocks used. 3. Detailed genotypes associated with each Fig. 4. Summary of mutant and RNAi lines used for each gene and associated phenotypes (Related to Supplementary Table 1) 5. Antibodies used in this work.
Supplementary Video 1
Nucleus positioning in live wild type larval fat bodies. In wild-type fat bodies, nucleus positioning was recorded in live whole larvae expressing His-GFP (nucleus, green) ubiquitously, and myr-RFP (plasma membrane, red) under the control of the fat body driver SPARC-Gal4. Movie records one hour of time-lapse imaging, sped up 60-fold. Note very little movement and maintenance of centroid positioning of nuclei.
Supplementary Video 2
Nucleus positioning defects after disruption of the fat body ncMTOC. In Msp300 RNAi fat bodies, nucleus positioning was recorded in live whole larvae expressing His-GFP (nucleus, green) ubiquitously, and myr-RFP (plasma membrane, red) under the control of the fat body driver SPARC-Gal4. Movie records one hour of time-lapse imaging, sped up 60-fold. Note very little nuclear movement and loss of nuclear centricity in these ncMTOC-disrupted fat body cells.
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Zheng, Y., Buchwalter, R.A., Zheng, C. et al. A perinuclear microtubule-organizing centre controls nuclear positioning and basement membrane secretion. Nat Cell Biol 22, 297–309 (2020). https://doi.org/10.1038/s41556-020-0470-7
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DOI: https://doi.org/10.1038/s41556-020-0470-7
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