Thorsten Kolb

Lamin A and lamin C form homodimers and coexist in higher complex forms both in the nucleoplasmic fraction and in the lamina of cultured human cells.

We have investigated and quantified the nuclear A-type lamin pool from human HeLa S3 suspension cells with respect to their distribution to detergent soluble and insoluble fractions. We devised a sequential extraction protocol and found that maximally 10% of A-type lamins are recovered in the soluble fraction. Notably, lamin C is enriched in low detergent fractions and only with 0.5% Nonidet P-40 lamin A and C are recovered in ratios nearly equivalent to those found in whole cell extracts and in the lamina fraction. Authentic nucleoplasmic proteins such as LAP2a, pRB and p53 are co-extracted to a large part together with the A-type lamins in these fractions. By sucrose density centrifugation we revealed that the majority of lamins co-sedimented with human IgG indicating they form rather small complexes in the range of dimers and slightly larger complexes. Some lamin A - but not lamin C - is obtained in addition in a much faster sedimenting fraction. Authentic nuclear proteins such as PCNA, p53 and LAP2a were found both in the light and the heavy sucrose fractions together with lamin A. Last but not least, immunoprecipitation experiments from both soluble fractions and from RIPA lysates of whole cells revealed that lamin A and lamin C do not form heterodimers but segregate practically completely. Correspondingly, immunofluorescence microscopy of formaldehyde-fixed cells clearly demonstrated that lamin A and C are localized at least in part to distinct patches within the lamina. Hence, the structural segregation of lamin A and C is indeed retained in the nuclear envelope to some extent too.

Microconstriction Arrays for High-Throughput Quantitative Measurements of Cell Mechanical Properties

We describe a method for quantifying the mechanical properties of cells in suspension with a microfluidic device consisting of a parallel array of micron-sized constrictions. Using a high-speed charge-coupled device camera, we measure the flow speed, cell deformation, and entry time into the constrictions of several hundred cells per minute during their passage through the device. From the flow speed and the occupation state of the microconstriction array with cells, the driving pressure across each constriction is continuously computed. Cell entry times into microconstrictions decrease with increased driving pressure and decreased cell size according to a power law. From this power-law relationship, the cell elasticity and fluidity can be estimated. When cells are treated with drugs that depolymerize or stabilize the cytoskeleton or the nucleus, elasticity and fluidity data from all treatments collapse onto a master curve. Power-law rheology and collapse onto a master curve are predicted by the theory of soft glassy materials and have been previously shown to describe the mechanical behavior of cells adhering to a substrate. Our finding that this theory also applies to cells in suspension provides the foundation for a quantitative high-throughput measurement of cell mechanical properties with microfluidic devices.

Induction of a Massive Endoplasmic Reticulum and Perinuclear Space Expansion by Expression of Lamin B Receptor Mutants and the Related Sterol Reductases TM7SF2 and DHCR7

We have studied the effect of disease-related lamin B receptor mutant proteins and of the related sterol reductases TM7SF2 and DHCR7 in human cultured cells. Our studies revealed that some of the tested protein variants massively interfered with regular organization of the endoplasmic reticulum, the nuclear envelope, and the nucleus.

Developmental regulation of linkers of the nucleoskeleton to the cytoskeleton during mouse postnatal retinogenesis.

Sun proteins and nesprins are two families of proteins whose direct interactions across the nuclear envelope provide for the core of linkers of the nucleoskeleton to the cytoskeleton (LINC complexes) that physically connect the nucleus interior to cytoskeletal networks. Whereas LINC complexes play essential roles in nuclear migration anchorage and underlie normal CNS development, the developmental regulation of their composition remains largely unknown. In this study, we examined the spatiotemporal expression of lamins, Sun proteins and nesprins during postnatal mouse retinal development. Whereas retinal precursor cells mostly express B-type lamins, Sun1, and high molecular weight isoforms of nesprins, post-mitotic retinal cells are characterized by a drastic downregulation of the latter, the expression of A-type lamins, and the strong induction of a specific isoform of nesprin1 late in retinal development. Importantly, our results emphasize different spatiotemporal expression for nesprin1 and nesprin2 and further suggest an important role for KASH-less isoforms of nesprin1 in the CNS. In conclusion, the transition from retinal precursor cells undergoing interkinetic nuclear migration to post-mitotic retinal cells undergoing nuclear translocation and/or anchorage is accompanied by a profound remodeling of LINC complexes composition. This remodeling may reflect different requirements of nuclear dynamics at different stages of CNS development.

Unbiased High-Precision Cell Mechanical Measurements with Microconstrictions

We describe a quantitative, high-precision, high-throughput method for measuring the mechanical properties of cells in suspension with a microfluidic device, and for relating cell mechanical responses to protein expression levels. Using a high-speed (750 fps) charge-coupled device camera, we measure the driving pressure Δp, maximum cell deformation εmax, and entry time tentry of cells in an array of microconstrictions. From these measurements, we estimate population averages of elastic modulus E and fluidity β (the power-law exponent of the cell deformation in response to a step change in pressure). We find that cell elasticity increases with increasing strain εmax according to E ∼ εmax, and with increasing pressure according to E ∼ Δp. Variable cell stress due to driving pressure fluctuations and variable cell strain due to cell size fluctuations therefore cause significant variability between measurements. To reduce measurement variability, we use a histogram matching method that selects and analyzes only those cells from different measurements that have experienced the same pressure and strain. With this method, we investigate the influence of measurement parameters on the resulting cell elastic modulus and fluidity. We find a small but significant softening of cells with increasing time after cell harvesting. Cells harvested from confluent cultures are softer compared to cells harvested from subconfluent cultures. Moreover, cell elastic modulus increases with decreasing concentration of the adhesion-reducing surfactant pluronic. Lastly, we simultaneously measure cell mechanics and fluorescence signals of cells that overexpress the GFP-tagged nuclear envelope protein lamin A. We find a dose-dependent increase in cell elastic modulus and decrease in cell fluidity with increasing lamin A levels. Together, our findings demonstrate that histogram matching of pressure, strain, and protein expression levels greatly reduces the variability between measurements and enables us to reproducibly detect small differences in cell mechanics.

Optofluidic rotation of living cells for single-cell tomography

Flow cytometry provides a high throughput, multi-dimensional analysis of cells flowing in suspension. In order to combine this feature with the ability to resolve detailed structures in 3D, we developed an optofluidic device that combines a microfluidic system with a dual beam trap. This allows for the rotation of single cells in a continuous flow, around an axis perpendicular to the imaging plane. The combination of both techniques enables the tomographic reconstruction of the 3D structure of the cell. In addition this method is capable to provide detailed 3D structural data for flow cytometry, as it improves the reconstructed z-resolution of a standard microscopy system to produce images with isotropic resolution in all three axes.

Genomic profiling of Acute lymphoblastic leukemia in ataxia telangiectasia patients reveals tight link between ATM mutations and chromothripsis

Recent developments in sequencing technologies led to the discovery of a novel form of genomic instability, termed chromothripsis. This catastrophic genomic event, involved in tumorigenesis, is characterized by tens to hundreds of simultaneously acquired locally clustered rearrangements on one chromosome. We hypothesized that leukemias developing in individuals with Ataxia Telangiectasia, who are born with two mutated copies of the ATM gene, an essential guardian of genome stability, would show a higher prevalence of chromothripsis due to the associated defect in DNA double-strand break repair. Using whole-genome sequencing, fluorescence in situ hybridization and RNA sequencing, we characterized the genomic landscape of Acute Lymphoblastic Leukemia (ALL) arising in patients with Ataxia Telangiectasia. We detected a high frequency of chromothriptic events in these tumors, specifically on acrocentric chromosomes, as compared with tumors from individuals with other types of DNA repair syndromes (27 cases total, 10 with Ataxia Telangiectasia). Our data suggest that the genomic landscape of Ataxia Telangiectasia ALL is clearly distinct from that of sporadic ALL. Mechanistically, short telomeres and compromised DNA damage response in cells of Ataxia Telangiectasia patients may be linked with frequent chromothripsis. Furthermore, we show that ATM loss is associated with increased chromothripsis prevalence in additional tumor entities.

Optomechanical measurement of the role of lamins in whole cell deformability

There is mounting evidence that the nuclear envelope, and particularly the lamina, plays a critical role in the mechanical and regulation properties of the cell and changes to the lamina can have implications for the physical properties of the whole cell. In this study we demonstrate that the optical stretcher can measure changes in the time-dependent mechanical properties of living cells with different levels of A-type lamin expression. Results from the optical stretcher shows a decrease in the deformability of cells as the levels of lamin A increases, for cells which grow both adherently and in suspension. Further detail can be probed by combining the optical stretcher with fluorescence microscopy to investigate the nuclear mechanical properties which show a larger decrease in deformability than for the whole cell.

Rotation of Optically Trapped Living Cells for Single-Cell Tomography

We use a dual-beam fibre trap integrated into a microfluidic system to hold single cells and rotate them about an axis perpendicular to the optical axis to give an isotropic tomographic image of the cell

Defective DNA damage repair leads to frequent catastrophic genomic events in murine and human tumors

Chromothripsis and chromoanasynthesis are catastrophic events leading to clustered genomic rearrangements. Whole-genome sequencing revealed frequent chromothripsis or chromoanasynthesis (n= 16/26) in brain tumors developing in mice deficient for factors involved in homologous-recombination-repair or non-homologous-end-joining. Catastrophic events were tightly linked to Myc/Mycn amplification, with increased DNA damage and inefficient apoptotic response already observable at early postnatal stages. Inhibition of repair processes and comparison of the mouse tumors with human medulloblastomas (n=68) and glioblastomas (n=32) identified chromothripsis as associated with MYC/MYCN gains and with DNA repair deficiencies, pointing towards therapeutic opportunities to target DNA repair defects in tumors with complex genomic rearrangements.