KIAA0319 influences cilia length, cell migration and mechanical cell-substrate interaction.

Following its association with dyslexia in multiple genetic studies, the KIAA0319 gene has been extensively investigated in different animal models but its function in neurodevelopment remains poorly understood. We developed the first human cellular knockout model for KIAA0319 in RPE1 retinal pigment epithelia cells via CRISPR-Cas9n to investigate its role in processes suggested but not confirmed in previous studies, including cilia formation and cell migration. We observed in the KIAA0319 knockout increased cilia length and accelerated cell migration. Using Elastic Resonator Interference Stress Microscopy (ERISM), we detected an increase in cellular force for the knockout cells that was restored by a rescue experiment. Combining ERISM and immunostaining we show that RPE1 cells exert highly dynamic, piconewton vertical pushing forces through actin-rich protrusions that are surrounded by vinculin-rich pulling sites. This protein arrangement and force pattern has previously been associated to podosomes in other cells. KIAA0319 depletion reduces the fraction of cells forming these actin-rich protrusions. Our results suggest an involvement of KIAA0319 in cilia biology and cell-substrate force regulation.

Real-time imaging of cellular forces using optical interference.

Important dynamic processes in mechanobiology remain elusive due to a lack of tools to image the small cellular forces at play with sufficient speed and throughput. Here, we introduce a fast, interference-based force imaging method that uses the illumination of an elastic deformable microcavity with two rapidly alternating wavelengths to map forces. We show real-time acquisition and processing of data, obtain images of mechanical activity while scanning across a cell culture, and investigate sub-second fluctuations of the piconewton forces exerted by macrophage podosomes. We also demonstrate force imaging of beating neonatal cardiomyocytes at 100 fps which reveals mechanical aspects of spontaneous oscillatory contraction waves in between the main contraction cycles. These examples illustrate the wider potential of our technique for monitoring cellular forces with high throughput and excellent temporal resolution.

Podocyte injury elicits loss and recovery of cellular forces.

In the healthy kidney, specialized cells called podocytes form a sophisticated blood filtration apparatus that allows excretion of wastes and excess fluid from the blood while preventing loss of proteins such as albumin. To operate effectively, this filter is under substantial hydrostatic mechanical pressure. Given their function, it is expected that the ability to apply mechanical force is crucial to the survival of podocytes. However, to date, podocyte mechanobiology remains poorly understood, largely because of a lack of experimental data on the forces involved. We perform quantitative, continuous, nondisruptive, and high-resolution measurements of the forces exerted by differentiated podocytes in real time using a recently introduced functional imaging modality for continuous force mapping. Using an accepted model for podocyte injury, we find that injured podocytes experience near-complete loss of cellular force transmission but that this loss of force is reversible under certain conditions. The observed changes in force correlate with F-actin rearrangement and reduced expression of podocyte-specific proteins. By introducing robust and high-throughput mechanical phenotyping and by demonstrating the significance of mechanical forces in podocyte injury, this research paves the way to a new level of understanding of the kidney. In addition, in an advance over established force mapping techniques, we integrate cellular force measurements with immunofluorescence and perform continuous long-term force measurements of a cell population. Hence, our approach has general applicability to a wide range of biomedical questions involving mechanical forces.

Analysis of the Precision, Robustness, and Speed of Elastic Resonator Interference Stress Microscopy.

Localization-microscopy-based methods are widely used to map the forces that cells apply to their substrates and to study important questions of cellular biomechanics. By contrast, elastic resonator interference stress microscopy (ERISM) uses an interference-based approach, which requires low light intensity and facilitates imaging of cellular forces with extreme precision (down to pN forces) and robustness (e.g., for continuous force monitoring over weeks). Here, the measurement trade-offs and numerical considerations required to optimize the performance of ERISM are described. The crucial parts of the fitting algorithm and the computational tools used to evaluate the data are explained in detail, and the precision and accuracy achievable with ERISM are analyzed. Additional features that can improve the robustness of ERISM further are discussed. The implementation of the analysis algorithm is verified with simulated test data and with experimental data. In addition, an approach to increase the acquisition speed of ERISM by a factor of four compared to the original implementation is described. In combination, these strategies allow us to measure the forces generated by a neural growth cone with high temporal resolution and continuously over several hours.

Long-term imaging of cellular forces with high precision by elastic resonator interference stress microscopy.

Cellular forces are crucial for many biological processes but current methods to image them have limitations with respect to data analysis, resolution and throughput. Here, we present a robust approach to measure mechanical cell-substrate interactions in diverse biological systems by interferometrically detecting deformations of an elastic micro-cavity. Elastic resonator interference stress microscopy (ERISM) yields stress maps with exceptional precision and large dynamic range (2 nm displacement resolution over a >1 µm range, translating into 1 pN force sensitivity). This enables investigation of minute vertical stresses (<1 Pa) involved in podosome protrusion, protein-specific cell-substrate interaction and amoeboid migration through spatial confinement in real time. ERISM requires no zero-force reference and avoids phototoxic effects, which facilitates force monitoring over multiple days and at high frame rates and eliminates the need to detach cells after measurements. This allows observation of slow processes such as differentiation and further investigation of cells, for example, by immunostaining.

Lasing in Live Mitotic and Non-Phagocytic Cells by Efficient Delivery of Microresonators.

Reliable methods to individually track large numbers of cells in real time are urgently needed to advance our understanding of important biological processes like cancer metastasis, neuronal network development and wound healing. It has recently been suggested to introduce microscopic whispering gallery mode lasers into the cytoplasm of cells and to use their characteristic, size-dependent emission spectrum as optical barcode but so far there is no evidence that this approach is generally applicable. Here, we describe a method that drastically improves intracellular delivery of resonators for several cell types, including mitotic and non-phagocytic cells. In addition, we characterize the influence of resonator size on the spectral characteristics of the emitted laser light and identify an optimum size range that facilitates tagging and tracking of thousands of cells simultaneously. Finally, we observe that the microresonators remain internalized by cells during cell division, which enables tagging several generations of cells.

Lasing within Live Cells Containing Intracellular Optical Microresonators for Barcode-Type Cell Tagging and Tracking.

We report on a laser that is fully embedded within a single live cell. By harnessing natural endocytosis of the cell, we introduce a fluorescent whispering gallery mode (WGM) microresonator into the cell cytoplasm. On pumping with nanojoule light pulses, green laser emission is generated inside the cells. Our approach can be applied to different cell types, and cells with microresonators remain viable for weeks under standard conditions. The characteristics of the lasing spectrum provide each cell with a barcode-type label which enables uniquely identifying and tracking individual migrating cells. Self-sustained lasing from cells paves the way to new forms of cell tracking, intracellular sensing, and adaptive imaging.