At the cutting edge: applications and perspectives of laser nanosurgery in cell biology

Ronchi, Paolo; Terjung, Stefan; Pepperkok, Rainer

doi:10.1515/hsz-2011-0237

Cited by 21 publications

(17 citation statements)

References 102 publications

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“…The first is the overall utility of tension across vinculin as a readout of whole-FA tension and traction, which as described above could be addressed with higherresolution measurements and a broader palette of optical sensors. Second, it would be informative to determine how SF tension distributions depend on the organization and anisotropy of the overall SF network, which can now be controlled to some extent by micropatterning (Ronchi et al, 2012;Théry, 2010). By dictating FA geometry and thus the length and orientation of intervening SFs, such measurements may also enable elucidation of length-tension relationships for SFs, which have thus far been difficult to clearly measure in living cells.…”

Section: Discussionmentioning

confidence: 99%

“…We and others have found femtosecond laser nanosurgery to be a powerful tool for measuring the contractile properties of single SFs in living cells (Colombelli et al, 2009;Colombelli et al, 2006;Kumar et al, 2006;Lele et al, 2006;Rauzi et al, 2008;Ronchi et al, 2012;Russell et al, 2009;Tanner et al, 2010). In this method, a cellular SF is visualized and irradiated with a femtosecond laser, resulting in SF scission and retraction.…”

Section: Introductionmentioning

confidence: 99%

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Vinculin tension distributions of individual stress fibers within cell-matrix adhesions

Chang

Kumar

2013

Journal of Cell Science

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SummaryActomyosin stress fibers (SFs) enable cells to exert traction on planar extracellular matrices (ECMs) by tensing focal adhesions (FAs) at the cell-ECM interface. Although it is widely appreciated that the spatial and temporal distribution of these tensile forces play key roles in polarity, motility, fate choice, and other defining cell behaviors, virtually nothing is known about how an individual SF quantitatively contributes to tensile loads borne by specific molecules within associated FAs. We address this key open question by using femtosecond laser ablation to sever single SFs in cells while tracking tension across vinculin using a molecular optical sensor. We show that disruption of a single SF reduces tension across vinculin in FAs located throughout the cell, with enriched vinculin tension reduction in FAs oriented parallel to the targeted SF. Remarkably, however, some subpopulations of FAs exhibit enhanced vinculin tension upon SF irradiation and undergo dramatic, unexpected transitions between tension-enhanced and tension-reduced states. These changes depend strongly on the location of the severed SF, consistent with our earlier finding that different SF pools are regulated by distinct myosin activators. We critically discuss the extent to which these measurements can be interpreted in terms of whole-FA tension and traction and propose a model that relates SF tension to adhesive loads and cell shape stability. These studies represent the most direct and highresolution intracellular measurements of SF contributions to tension on specific FA proteins to date and offer a new paradigm for investigating regulation of adhesive complexes by cytoskeletal force.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Vinculin tension distributions of individual stress fibers within cell-matrix adhesions

Chang

Kumar

2013

Journal of Cell Science

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“…More specifically, femtosecond laser-induced optical breakdown in water and aqueous media enables to perform highly precise surgery on cells [1][2][3] and within transparent biological tissues [4][5][6][7][8]. The dependence of optical breakdown thresholds on laser parameters provides information about breakdown mechanisms such as the interplay of strong-field ionization (SFI) and avalanche ionization (AI) [9][10][11][12][13][14], as well as about the band structure of the breakdown medium and the mechanisms of breakdown initiation [15,16].…”

Section: Introductionmentioning

confidence: 99%

Wavelength dependence of femtosecond laser-induced breakdown in water and implications for laser surgery

et al. 2016

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The wavelength dependence of the threshold for femtosecond optical breakdown in water provides information on the interplay of multiphoton, tunneling and avalanche ionization, and is of interest for parameter selection in laser surgery. We measured the bubble threshold from UV to near-IR wavelengths and found a continuous decrease of the irradiance threshold with increasing wavelength λ. Results are compared to the predictions of a numerical model that assumes a bandgap of 9.5 eV and considers the existence of a separate initiation channel via excitation of valence band electrons into a solvated state followed by rapid upconversion into the conduction band. Fits to experimental data yield an electron collision time of ≈ 1 fs, and an estimate for the capacity of the initiation channel. Using that collision time, the breakdown dynamics was explored up to λ = 2 µm. The irradiance threshold first continues to decrease but levels out for wavelengths longer than 1.3 µm. This opens promising perspectives for laser surgery at wavelengths around 1.3 µm and 1.7 µm, which are attractive because of their large penetration depth into scattering tissues.

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“…To address this question, we made use of a recently developed technique to deplete the Golgi complex from living cells by laser nanosurgery and monitor its subsequent biogenesis (Tängemo et al, 2011;Ronchi et al, 2012;Ronchi and Pepperkok, 2013). This approach has provided direct evidence Fig.…”

Section: Introductionmentioning

confidence: 99%

Positive feedback between golgi membranes, microtubules and ER-exit sites directs golgi de novo biogenesis

Ronchi

Tischer²,

Acehan

et al. 2014

Journal of Cell Science

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The Golgi complex is the central organelle of the secretory pathway. It undergoes dynamic changes during the cell cycle, but how it acquires and maintains its complex structure is unclear. To address this question, we have used laser nanosurgery to deplete BSC1 cells of the Golgi complex and have monitored its biogenesis by quantitative time-lapse microscopy and correlative electron microscopy. After Golgi depletion, endoplasmic reticulum (ER) export is inhibited and the number of ER exit sites (ERES) is reduced and does not increase for several hours. Occasional fusion of small post-ER carriers to form the first larger structures triggers a rapid and drastic growth of Golgi precursors, due to the capacity of these structures to attract more carriers by microtubule nucleation and to stimulate ERES biogenesis. Increasing the chances of post-ER carrier fusion close to ERES by depolymerizing microtubules results in the acceleration of Golgi and ERES biogenesis. Taken together, on the basis of our results, we propose a self-organizing principle of the early secretory pathway that integrates Golgi biogenesis, ERES biogenesis and the organization of the microtubule network by positive-feedback loops.

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At the cutting edge: applications and perspectives of laser nanosurgery in cell biology

Cited by 21 publications

References 102 publications

Vinculin tension distributions of individual stress fibers within cell-matrix adhesions

Vinculin tension distributions of individual stress fibers within cell-matrix adhesions

Wavelength dependence of femtosecond laser-induced breakdown in water and implications for laser surgery

Positive feedback between golgi membranes, microtubules and ER-exit sites directs golgi de novo biogenesis

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