Centrifugal Displacement of Nuclei in Adherent Cells to Study LINC Complex-Dependent Mechanisms of Homeostatic Nuclear Positioning

2018 ◽  
pp. 91-100
Author(s):  
Ruijun Zhu ◽  
Gregg G. Gundersen
Keyword(s):  
Adherent Cells ◽  
Linc Complex ◽  
Nuclear Positioning

scholarly journals Centrifugal Displacement of Nuclei Reveals Multiple LINC Complex Mechanisms for Homeostatic Nuclear Positioning

2017 ◽  
Vol 27 (20) ◽  
pp. 3097-3110.e5 ◽  
Author(s):  
Ruijun Zhu ◽  
Susumu Antoku ◽  
Gregg G. Gundersen
Keyword(s):  
Linc Complex ◽  
Nuclear Positioning ◽  
Complex Mechanisms

scholarly journals Nucleus-dependent sarcomere assembly is mediated by the LINC complex

2016 ◽  
Vol 27 (15) ◽  
pp. 2351-2359 ◽  
Author(s):  
Alexander L. Auld ◽  
Eric S. Folker
Keyword(s):  
Muscle Cells ◽  
Linc Complex ◽  
Nuclear Positioning ◽  
Nuclear Position ◽  
Early Stages ◽  
Cytoplasmic Face ◽  
Sarcomere Proteins ◽  
The Many ◽  
Sarcomere Assembly ◽  
The Relationship

Two defining characteristics of muscle cells are the many precisely positioned nuclei and the linearly arranged sarcomeres, yet the relationship between these two features is not known. We show that nuclear positioning precedes sarcomere formation. Furthermore, ZASP-GFP, a Z-line protein, colocalizes with F-actin in puncta at the cytoplasmic face of nuclei before sarcomere assembly. In embryos with mispositioned nuclei, ZASP-GFP is still recruited to the nuclei before its incorporation into sarcomeres. Furthermore, the first sarcomeres appear in positions close to the nuclei, regardless of nuclear position. These data suggest that the interaction between sarcomere proteins and nuclei is not dependent on properly positioned nuclei. Mechanistically, ZASP-GFP localization to the cytoplasmic face of the nucleus did require the linker of nucleoskeleton and cytoskeleton (LINC) complex. Muscle-specific depletion of klarsicht (nesprin) or klariod (SUN) blocked the recruitment of ZASP-GFP to the nucleus during the early stages of sarcomere assembly. As a result, sarcomeres were poorly formed and the general myofibril network was less stable, incomplete, and/or torn. These data suggest that the nucleus, through the LINC complex, is crucial for the proper assembly and stability of the sarcomere network.

scholarly journals Nesprin 1α2 is essential for mouse postnatal viability and nuclear positioning in skeletal muscle

2017 ◽  
Vol 216 (7) ◽  
pp. 1915-1924 ◽  
Author(s):  
Matthew J. Stroud ◽  
Wei Feng ◽  
Jianlin Zhang ◽  
Jennifer Veevers ◽  
Xi Fang ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Knockout Mice ◽  
Muscle Function ◽  
Actin Binding ◽  
Muscle Dysfunction ◽  
Linc Complex ◽  
Nuclear Positioning ◽  
Skeletal Muscle Function ◽  
Binding Domains ◽  
A Cell

The position of the nucleus in a cell is controlled by interactions between the linker of nucleoskeleton and cytoskeleton (LINC) complex and the cytoskeleton. Defects in nuclear positioning and abnormal aggregation of nuclei occur in many muscle diseases and correlate with muscle dysfunction. Nesprin 1, which includes multiple isoforms, is an integral component of the LINC complex, critical for nuclear positioning and anchorage in skeletal muscle, and is thought to provide an essential link between nuclei and actin. However, previous studies have yet to identify which isoform is responsible. To elucidate this, we generated a series of nesprin 1 mutant mice. We showed that the actin-binding domains of nesprin 1 were dispensable, whereas nesprin 1α2, which lacks actin-binding domains, was crucial for postnatal viability, nuclear positioning, and skeletal muscle function. Furthermore, we revealed that kinesin 1 was displaced in fibers of nesprin 1α2–knockout mice, suggesting that this interaction may play an important role in positioning of myonuclei and functional skeletal muscle.

scholarly journals Ctdnep1 and Eps8L2 regulate dorsal actin cables for nuclear positioning during cell migration

2020 ◽  
Author(s):  
Francisco J. Calero-Cuenca ◽  
Daniel S. Osorio ◽  
Sreerama Chaitanya Sridhara ◽  
Yue Jiao ◽  
Jheimmy Diaz ◽  
...  
Keyword(s):  
Cell Migration ◽  
Nuclear Envelope ◽  
Leading Edge ◽  
Cell Types ◽  
Linc Complex ◽  
Nuclear Positioning ◽  
Physiological Functions ◽  
Actin Cables ◽  
Different Cell Types

SummaryCells actively position their nuclei within the cytoplasm for multiple cellular and physiological functions. Different cell types position their nuclei away from the leading edge to migrate properly. In migrating fibroblasts, nuclear positioning is driven by dorsal actin cables connected to the nuclear envelope by the LINC complex on Transmembrane Actin-associated Nuclear (TAN) lines. How dorsal actin cables are organized to form TAN lines is unknown. Here, we report a role for Ctdnep1/Dullard, a nuclear envelope phosphatase, and the actin regulator Eps8L2, on nuclear positioning. We demonstrate that Ctdnep1 and Eps8L2 directly interact to regulate the formation and thickness of dorsal actin cables required for TAN lines engagement for nuclear positioning. Our work establishes a novel mechanism to locally regulate actin at the nuclear envelope for nuclear positioning.

scholarly journals Linker of nucleoskeleton and cytoskeleton (LINC) complex-mediated actin-dependent nuclear positioning orients centrosomes in migrating myoblasts

Nucleus ◽  
2015 ◽  
Vol 6 (1) ◽  
pp. 77-88 ◽  
Author(s):  
Wakam Chang ◽  
Susumu Antoku ◽  
Cecilia Östlund ◽  
Howard J Worman ◽  
Gregg G Gundersen
Keyword(s):  
Linc Complex ◽  
Nuclear Positioning

scholarly journals Chain reaction: LINC complexes and nuclear positioning

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 136 ◽  
Author(s):  
Brian Burke
Keyword(s):  
Nuclear Membrane ◽  
Nuclear Periphery ◽  
Cell Types ◽  
Linc Complex ◽  
Nuclear Movement ◽  
Nuclear Positioning ◽  
Cell Architecture ◽  
Nuclear Location ◽  
Complex Regulation ◽  
Nuclear Structures

Nuclear positioning plays an essential role in defining cell architecture and behaviour in both development and disease, and nuclear location frequently adjusts according to internal and external cues. For instance, during periods of migration in many cell types, the nucleus may be actively repositioned behind the microtubule-organising centre. Nuclear movement, for the most part, is dependent upon coupling of the cytoskeleton to the nuclear periphery. This is accomplished largely through SUN and KASH domain proteins, which together assemble to form LINC (linker of the nucleoskeleton and cytoskeleton) complexes spanning the nuclear envelope. SUN proteins of the inner nuclear membrane provide a connection to nuclear structures while acting as a tether for outer nuclear membrane KASH proteins. The latter contain binding sites for diverse cytoskeletal components. Recent publications highlight new aspects of LINC complex regulation revealing that the interplay between SUN and KASH partners can strongly influence how the nucleus functionally engages with different branches of the cytoskeleton.

scholarly journals UnLINCing the nuclear envelope: towards an understanding of the physiological significance of nuclear positioning

2011 ◽  
Vol 39 (6) ◽  
pp. 1790-1794 ◽  
Author(s):  
David Razafsky ◽  
Shulun Zang ◽  
Didier Hodzic
Keyword(s):  
Nuclear Envelope ◽  
Molecular Motors ◽  
Developmental Regulation ◽  
Linc Complex ◽  
Nuclear Positioning ◽  
Nuclear Dynamics ◽  
Physiological Relevance ◽  
Different Types ◽  
Cytoskeletal Elements

Appropriate tissue morphogenesis strictly requires the developmental regulation of different types of nuclear movements. LINC (linker of nucleoskeleton and cytoskeleton) complexes are macromolecular scaffolds that span the nuclear envelope and physically connect the nuclear interior to different cytoskeletal elements and molecular motors, thereby playing essential roles in nucleokinesis. Recent studies dedicated to the in vivo disruption of LINC complexes not only confirmed their widespread role in nuclear dynamics, but also led to a vigorous regain of interest in the physiological relevance of nuclear positioning within cells and syncitia. In the present paper, we review the results of LINC complex disruption in vivo across different organisms and the potential implications of observed phenotypes in human diseases.

3-D organization of fibrinogen receptors using computer reconstruction and whole-mount microscopy

1988 ◽  
Vol 46 ◽  
pp. 30-31
Author(s):  
E. T. O'Toole ◽  
R. R. Hantgan ◽  
J. C. Lewis
Keyword(s):  
Whole Blood ◽  
Colloidal Gold ◽  
Blood Platelets ◽  
Cell Contact ◽  
Computer Assisted ◽  
Adherent Cells ◽  
Differential Centrifugation ◽  
Computer Reconstruction ◽  
Sds Page ◽  
Whole Mount

Thrombocytes (TC), the avian equivalent of blood platelets, support hemostasis by aggregating at sites of injury. Studies in our lab suggested that fibrinogen (fib) is a requisite cofactor for TC aggregation but operates by an undefined mechanism. To study the interaction of fib with TC and to identify fib receptors on cells, fib was purified from pigeon plasma, conjugated to colloidal gold and used both to facilitate aggregation and as a receptor probe. Described is the application of computer assisted reconstruction and stereo whole mount microscopy to visualize the 3-D organization of fib receptors at sites of cell contact in TC aggregates and on adherent cells.Pigeon TC were obtained from citrated whole blood by differential centrifugation, washed with Ca++ free Hank's balanced salts containing 0.3% EDTA (pH 6.5) and resuspended in Ca++ free Hank's. Pigeon fib was isolated by precipitation with PEG-1000 and the purity assessed by SDS-PAGE. Fib was conjugated to 25nm colloidal gold by vortexing and the conjugates used as the ligand to identify fib receptors.

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