Nuclear mechanics in laminopathies

Investigator: Jan Lammerding
Meinig School of Biomedical Engineering, Cornell University

Investigator's profile

Lammerding laboratory is a leader in studies on mechanotransduction - a process by which cells sense mechanical forces and deformations, and respond through cytoskeletal reorganization, biochemical signaling, and specific biological functions. His lab has demonstrated that mutations in the LMNA gene encoding the Lamin A/C proteins lead to impaired nuclear mechanics (see Fig. 1), which is responsible for defective mechanotransduction [1] and hence for the on start of a number of human genetic diseases including muscular dystrophies, cardiomyopathies, lipodystrophies and progerioid phenotypes.

Significance

Figure 1: (a) Wide-type and (b) lamin A/C deficient nuclei under similar substrate strain. (c) The lamin-deficient nucleus exhibits significantly greater strain.

Beside laminopathies, nuclear mechanics is also altered in cancer cells and plays an important role during cancer metastasis. Rheological measurements generally involve applying precisely controlled stresses and monitoring the resultant strains. When measuring nuclear mechanics, one challenge with the most common approaches such as micropipette aspiration, atomic force microscopy (AFM) indentation, and optical / magnetic tweezers is that the mechanical loading / stimulation can often be only indirect since the nucleus is surrounded by cell cytoskeleton. Lammerding Lab has also developed novel assays for nuclear mechanics including cellular strain assay in which cells are cultured on fibronectin-coated silicone membranes and subjected to well-defined uniaxial or biaxial strain, and microneedle manipulation assay that applies precisely controlled cytoskeletal strain at a defined distance from the nucleus while simultaneously imaging induced nuclear and cytoskeletal deformations [2]. Despite these efforts, the exact mechanisms underlying laminopathies are not well understood due technology limitations. LBRC aims to develop novel biomechanical assays to study, in collabortion with Lammerding Lab, the complex interplay between nuclear envelope mechanics and surrounding cytoskeleton especially in the context of laminopathies.

Approach

Figure 2: Biomechanical assay based on 3-D resolved interferometric measurement of nanometer scale nuclear membrane fluctuations.

In the past, LBRC has successfully developed and applied transmission-type quantitative phase microscopy-based optical assays [3] for non-contact study of red blood cell biomechanics [4,5]. 3-D resolved interferometric biomechanical assays, however, are needed to study biomechanical properties of complex eukaryotic cells. In the recent past, LBRC has also made major progress in 3-D resolved interferometric imaging assays [6]; however, the achieved phase measurement sensitivity was not sufficient to support this collaboration. The development of common-path 3-D resolved optical imaging assay will provide desired axial motion sensitivity to quantify cellular biophysics with high spatial and temporal resolution. Furthermore, novel plasmonic nanopillar platform can provide nanometric deformation as well as real-time vibrational spectroscopic measurement in live functioning cells to provide insights into how different cells recognize spatial and biochemical domains, at the sub-micron scale, correlating biochemical changes to the mechanical deformations / cell mechanics.

Research plans

Figure 3: Biomechanical assay based on nanopillar array.

The LBRC is developing a multi-spot scanning confocal reflection phase microscope that will feature the necessary features such as 1 micron depth-sectioning, fast (0.1-1kHz) frame rate, and high (< 1nm axial motion) measurement sensitivity (TRD2.1). The developed technology will enable measuring sub-nanometer plasma and nucleic membrane fluctuations, which will be further used with an appropriate model to extract nucleic as well as overall rhelogical properties of the biological cell under observation. In addition, LBRC will develop a complementary approach based on plasmonic nanoparticle-coated vertical nanopillar array (TRD3.2) with single-molecule detection sensitivity and surface selectivity (Fig. 3). The proposed technology will enable non-invasive subcellular perturbations and simultaneous nanoscale monitoring of events at the cell surface. In addition, the plasmonic nanoparticle nanopillar array will also enable simultaneous detection of membrane biomarkers responsible for cell signaling, migration, and proliferation.

Summary

The LBRC will develop a highly sensitive non-invasive interferometric 3D biomechanical assay to study nuclear mechanics of Lamin A/C deficient cells. In addition, the center will also develop a complementary approach based on nanopillar array to quantify sub-cellular responses to controlled mechanical stimuli. Successful development of these assays will not only help study laminopathies, but will also enable studying the role of nulcear mechanics in cancer metastasis.

References

  1. "Nuclear lamin A/C deficiency induces defects in cell mechanics, polarization, and migration," Biophysical Journal, 2007. [ Link ]
  2. "Assays to measure nuclear mechanics in interphase cells," Current protocols in cell biology, 2012. [ Link ]
  3. "Diffraction phase microscopy for quantifying cell structure and dynamics," Optics Letters, 2006. [ Pubmed ]
  4. "Optical measurement of cell membrane tension," PRL, vol. 97, Issue 21, pp. Optical measurement of cell membrane tension, Nov 2006. [ Pubmed ]
  5. "Refractive index maps and membrane dynamics of human red blood cells parasitized by Plasmodium falciparum," PNAS, vol. 105, Issue 37, pp. 13730-13735, Sep 2008. [ Pubmed ]
  6. "Dynamic speckle illumination wide-field reflection phase microscopy," Optics Letters, 2006. [ Pubmed ]