W&M ScholarWorks Arts & Sciences Articles Arts and Sciences 2017 Lamin A and microtubules collaborate to maintain nuclear morphology Zeshan Tariq [email protected] Haoyue Zhang Alexander Chia-Liu Yang Shen Yantenew Gete See next page for additional authors Follow this and additional works at: https://scholarworks.wm.edu/aspubs Recommended Citation Tariq, Zeshan; Zhang, Haoyue; Chia-Liu, Alexander; Shen, Yang; Gete, Yantenew; Xiong, Zheng-Mei; Tocheny, Claire; Campanello, Leonard; Wu, Di; Losert, Wolfgang; and Cao, Kan, Lamin A and microtubules collaborate to maintain nuclear morphology (2017). 10.1080/19491034.2017.1320460 This Article is brought to you for free and open access by the Arts and Sciences at W&M ScholarWorks. It has been accepted for inclusion in Arts & Sciences Articles by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. Authors Zeshan Tariq, Haoyue Zhang, Alexander Chia-Liu, Yang Shen, Yantenew Gete, Zheng-Mei Xiong, Claire Tocheny, Leonard Campanello, Di Wu, Wolfgang Losert, and Kan Cao This article is available at W&M ScholarWorks: https://scholarworks.wm.edu/aspubs/265 NUCLEUS 2017, VOL. 8, NO. 4, 433–446 https://doi.org/10.1080/19491034.2017.1320460 ORIGINAL RESEARCH Lamin A and microtubules collaborate to maintain nuclear morphology Zeshan Tariqa, Haoyue Zhanga, Alexander Chia-Liub, Yang Shenb, Yantenew Getea, Zheng-Mei Xiong a, Claire Tochenyc, Leonard Campanellob,DiWua, Wolfgang Losertb, and Kan Caoa aDepartment of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA; bDepartment of Physics, University of Maryland, College Park, MD, USA; cDepartment of Biology, The College of William and Mary, Williamsburg, VA, USA ABSTRACT ARTICLE HISTORY Lamin A (LA) is a critical structural component of the nuclear lamina. Mutations within the LA Received 7 November 2016 gene (LMNA) lead to several human disorders, most striking of which is Hutchinson-Gilford Revised 30 March 2017 Progeria Syndrome (HGPS), a premature aging disorder. HGPS cells are best characterized by Accepted 12 April 2017 an abnormal nuclear morphology known as nuclear blebbing, which arises due to the KEYWORDS accumulation of progerin, a dominant mutant form of LA. The microtubule (MT) network is HGPS; lamin A; nuclear knowntomediatechangesinnuclearmorphology in the context of specificeventssuchas shape; microbutuble; mitosis, cell polarization, nucleus positioning and cellular migration. What is less understood is progerin the role of the microtubule network in determining nuclear morphology during interphase. In this study, we elucidate the role of the cytoskeletoninregulationandmisregulationofnuclear morphology through perturbations of both the lamina and the microtubule network. We found that LA knockout cells exhibit a crescent shape morphology associated with the microtubule-organizing center. Furthermore, this crescent shape ameliorates upon treatment with MT drugs, Nocodazole or Taxol. Expression of progerin, in LA knockout cells also rescues the crescent shape, although the response to Nocodazole or Taxol treatment is altered in comparison to cells expressing LA. Together these results describe a collaborative effort between LA and the MT network to maintain nuclear morphology. Introduction of age.5,12-14 HGPS arises due to the accumulation of a Lamin A (LA), a type V intermediate filament encoded mutant LA isoform termed progerin which anchors to by the LMNA gene, is a key component of the nuclear the nuclear membrane and leads to thickening of the lamina.1 The lamina supports the nuclear envelope, nuclear lamina, loss of heterochromatin, alterations in allowing the nucleus to resist mechanical perturba- histone methylation, gene misregulation, and genomic tions.2,3 LA directly interacts with chromosomes and instability.4,6,15-21 Most Evidently, the accumulation of nuclear regulatory proteins, implicating the nuclear progerin results in an abnormal nuclear morphology lamina in key cellular processes such as apoptosis, termed nuclear blebbing.16 Nuclear blebbing appears chromatin organization, and gene expression.4-9 to drive the pathology of HGPS, as treatments that Mutations in the LMNA gene have been associated improve blebbing have been associated with amelio- with a heterogeneous, rare group of hereditary dis- rated HGPS cellular phenotypes.10,22-28 A change in eases collectively termed laminopathies that include nuclear shape is also suggested to be a hallmark of Emery–Dreifuss Muscular Dystrophy, Dunnigan-type lamin mutations that lead to other laminopathies.29 familial partial lipodystrophy, dilated cardiomyopa- The nucleus is not an isolated system. Prior studies thy, and Hutchinson-Gilford Progeria Syndrome have shown that the cytoskeleton directly influences (HGPS).1,5,10,11 Among these laminopathies, HGPS nuclear shape in fundamental cellular processes.30,31 has been extensively studied due to its striking clinical The most extreme example is the Microtubule (MT) phenotypes including osteoporosis, loss of subcutane- facilitated nuclear breakdown during mitosis.32 Fur- ous fat, alopecia, and joint stiffening after 12 months thermore, during cell migration, a filamentous actin CONTACT Kan Cao [email protected] Department of Cell Biology and Molecular Genetics, 2114 Bioscience Research Building, College Park, MD 20742, USA. Supplemental data for this article can be accessed on the publisher’s website. © 2017 Taylor & Francis 434 Z. TARIQ ET AL. structure known as the actin cap compresses and sup- To accurately and quantitatively determine the ports the nucleus.33 Studies investigating granulopoiesis nuclear shape, we applied a described previously found that the MT network mediates nuclear lobula- method for nuclear measurements.45,46 This program tion.34 Other studies have focused on MT influences reports nuclear morphology as a measure of local during migration/positioning. In Drosophila follicle invaginations typically associated with blebs and cells, individual MT fibers push the nucleus creating nuclear deformations. Mean negative curvature local indentations resulting in nuclear “wriggling”.35 In (MNC), one of the reported measures, is defined as Drosophila oocytes, the dorsal-ventral axis is polarized the absolute value of the average negative curvature by nucleus migration. This process is mediated by the (negative when measured relative to the center of the MTOC which pushes the nucleus causing an indenta- nucleus) excluding all positive curvature values, i.e. all tion.36 A direct connection between the nuclear lamina regions where the nucleus bulges outward. While it is and the cytoskeleton has been established through counterintuitive to not measure outward bulges, our LINC complexes, transmembrane proteins that span previous work clearly showed that blebs are best iden- the nuclear membrane and bind to both.37 These stud- tified from their surrounding inward invagina- ies, as well as others, have focused on event specific tions.45,46 Indeed, nuclei we would identify as more microtubule-nucleus interactions.33,34,37-41 In compari- abnormal by visual inspection exhibit higher MNC. son, relatively little is known about how the equilibrium Using lamin B1 (LB1) immunofluorescence staining, nuclear morphology is affected by the cytoskeleton, we observed that LA¡/¡ cells more frequently exhib- especially in laminopathies. Interestingly, NAT10 ited a crescent shaped nuclear morphology compared inhibitor, remodelin has been associated with improved with LAC/C cells (Fig. 1A). We then counted over 100 nuclear morphology and cellular fitness in HGPS cells randomly selected nuclei and found that the frequency through a mechanism targeting the MT organizing cen- of crescent-shaped nuclei was significantly higher in ter (MTOC),42 which suggests a role for the cytoskele- LA¡/¡ cells than LAC/C cells (Fig. 1B). The crescent ton in the nuclear blebbing phenotype in HGPS. shape of the nucleus was associated with gamma tubu- In this study, we investigate the interplay between lin localization to the arc of the crescent (Fig. 1C and microtubules and the nuclear lamina to elucidate the D), suggesting that the MTOC may be involved in this role of the cytoskeleton in maintaining normal nuclear nuclear abnormality in the absence of LA. morphology. We find that the MT network in the Nuclear shape analysis showed that LA¡/¡ nuclei presence of LA is necessary for appropriate nuclear exhibited increased mean negative curvature (Fig. 1E) morphology. The absence of LA led to MT-driven and reduced nuclear area (Fig. 1F), suggesting that nuclear abnormalities. Furthermore, the presence of LA¡/¡ nuclei displayed worsened morphology. The progerin led to the altered MT-nucleus interactions. differences were significant enough allowing for Together, these results suggest that normal nuclear robust identification of a boundary between the 2 con- shape is dependent upon balanced interactions ditions (Fig. 1G). When using all the 5 metrics of between both cytoskeletal and lamin networks. nuclear shape (i.e., MNC, area, eccentricity, solidity, and tortuosity) for classification, the classification Results accuracy is quite high at 88%, with a 10% likelihood of classifying a single LAC/C cell as LA¡/¡, and a 13% Loss of LA results in the MTOC-associated crescent- chance of classifying a single LA¡/¡ cell as LAC/C. shaped nuclei These results show that LA is necessary for appropri- The MT network is dynamic, with MTs growing and ate nuclear morphology, and further suggests that the pushing,