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This volume describes research methodologies and approaches used to study the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex and its cellular functions. Chapters detail structural and biochemical analysis of LINC complexes, mechanical aspects of the LINC complex, analysis of the LINC complex in model systems and development, and LINC complex in mammalian tissue, organs, and disease. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and cutting-edge, The LINC Complex: Methods and Protocols aims to ensure successful results in the further study of this vital field.
List of contents
EM Tomography of LINC Complexes.- Recombinant Purification of the Periplasmic Portion of the LINC Complex.- Analysis of High Molecular Weight Isoforms of Nesprin-1 and Nesprin-2 with Vertical Agarose Gel Electrophoresis.- Analysis of Nesprin-2 Interaction with its Binding Partners and Actin.- Interactions of Nesprin-4-containing LINC Complexes in Outer Hair Cells Explored by BioID.- Using Nesprin Tension Sensors to Measure Force on the LINC Complex.- Analyzing Mechanotransduction through the LINC Complex in Isolated Nuclei.- Direct Force Probe for Nuclear Mechanics.- Centrifugal Displacement of Nuclei in Adherent Cells to Study LINC Complex Dependent Mechanisms of Homeostatic Nuclear Positioning.- Assembly and Use of a Microfluidic Device to Study Cell Migration in Confined Environments.- Investigating LINC Complex Protein Homo-oligomerization in the Nuclear Envelopes of Living Cells Using Fluorescence Fluctuation Spectroscopy.- Functional Analysis of the Yeast LINC Complex Using Fluctuation Spectroscopy and Super Resolution Imaging.- Genetic Analysis of Nuclear Migration and Anchorage to Study LINC Complexes During Development of Caenorhabditis elegans.- High-resolution Imaging Methods to Analyze LINC Complex Function During Drosophila Muscle Development.- Computational Methods for Studying the Plant Nucleus.- Investigation of Nuclear Periphery Protein Interactions in Plants Using the Membrane Yeast Two-Hybrid (MbY2H) System.- Immunolabeling Protocols for Studying Meiosis in Plant Mutants Defective for Nuclear Envelope Components.- Generation and Analysis of Striated Muscle Selective LINC Complex Protein Mutant Mice.- An in vitro System to Measure the Positioning, Stiffness and Rupture of the Nucleus in Skeletal Muscle.- Functional Analysis of LINC Complexes in Skin.- Detection of SUN1 Splicing Variants at the mRNA and Protein Levels in Cancer.- Next-generation Sequencing and Mutational Analysis: Implications for Genes Encoding LINC Complex Proteins
