tweezercalib 2.1: Faster version of MatLab package for precise calibration of optical tweezers

Cells can interact with their surroundings via filopodia, which are membrane protrusions that extend beyond the cell body. Filopodia are essential during dynamic cellular processes like motility, invasion, and cell-cell communication. Filopodia contain crosslinked actin filaments, attached to the surrounding cell membrane via protein linkers such as integrins. These actin filaments are thought to play a pivotal role in force transduction, bending, and rotation. We investigated whether, and how, actin within filopodia is responsible for filopodia dynamics by conducting simultaneous force spectroscopy and confocal imaging of F-actin in membrane protrusions. The actin shaft was observed to periodically undergo helical coiling and rotational motion, which occurred simultaneously with retrograde movement of actin inside the filopodium. The cells were found to retract beads attached to the filopodial tip, and retraction was found to correlate with rotation and coiling of the actin shaft. These results suggest a previously unidentified mechanism by which a cell can use rotation of the filopodial actin shaft to induce coiling and hence axial shortening of the filopodial actin bundle.membrane-cytoskeleton interactions | membrane nanotubes | filopodial retrograde flow | helical buckling | filopodia rotation T ubular membrane remodeling driven by the actin cytoskeleton plays a major role in both pathogenesis and in a healthy immune response. For instance, invadopodia, podosomes, and filopodia are crucial for invasion and migration of cells (1). Filopodia are thin (100 to 300 nm), tube-like, actin-rich structures that function as "antennae" or "tentacles" that cells use to probe and interact with their microenvironment (2-4). Such structures have been studied in vitro using model systems where the point-like 3D contacts with the extracellular matrix (ECM) have been mimicked by using optically trapped dielectric particles, functionalized with relevant ligands. These model systems allow for mechanical and visual control over filopodial dynamics and have significantly advanced the understanding of cellular mechanosensing (5).Extraction of membrane tubes, using optical trapping, has been used to investigate mechanical properties of the membrane-cytoskeleton system (6-10), or membrane cholesterol content (11), and has revealed important insight into the mechanism that peripheral proteins use to shape membranes (12). Motivated by the pivotal role of F-actin in the mechanical behavior of filopodia and other cellular protrusions, special focus has been on revealing the presence of F-actin within extracted membrane tubes (1, 13-16). However, apart from a single study (9), fluorescent visualization of the F-actin was achieved by staining and fixation of the cells, and literature contains conflicting results regarding the presence or absence of actin in membrane tubes pulled from living cells (8,11,17).Filopodia in living cells have the ability to rotate and bend by a so-far unknown mechanism (13,(18)(19)(20). For instance, filopodia ...

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“…26. More details concerning the experimental setup and Matlab analysis (47) are provided in SI Appendix, SI Text.…”

Section: Methodsmentioning

confidence: 99%

Helical buckling of actin inside filopodia generates traction

Leijnse

Oddershede

Bendix³

2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Recently, an all-optical force-clamping method was introduced that operates without an electronic feedback, thus increasing significantly the bandwidth of the force and displacement measurements [304]. Fast Matlab-based routines for precise calibration of position and force measurements with OT have been developed and are freely available [305][306][307].Force-measuring OT in their various modifications have found numerous successful applications in characterizing the structure of and force interactions between molecular components of living cells -molecular motors carrying out intracellular transport (myosin-actin and kinesin/dyneinmicrotubule systems), DNA and RNA polymerases, biopolymers (DNA, RNA, actin, microtubules), and ligandreceptor systems (e.g. antibody-antigen) (see [11][12][13][14][216][217][218] for a comprehensive review).…”

mentioning

confidence: 99%

Light at work: The use of optical forces for particle manipulation, sorting, and analysis

2008

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We review the combinations of optical micro-manipulation with other techniques and their classical and emerging applications to non-contact optical separation and sorting of micro- and nanoparticle suspensions, compositional and structural analysis of specimens, and quantification of force interactions at the microscopic scale. The review aims at inspiring researchers, especially those working outside the optical micro-manipulation field, to find new and interesting applications of these methods.

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“…Using the position sensitive diode the time series of the sphere was recorded and used for a calibration routine which returned the corner frequency f c , the trap stiffness κ, and the conversion factor β. shows the corresponding power spectrum. Here, the light grey part is fitted by a Lorentzian function (black full line) which takes into account the filtering effect of the photodiode [23] and aliasing [19] using programs described above [20]. Though the system is subject to low-frequency drift, this does not show neither in the time series, nor in the histogram, nor in the power spectrum, not even when compared to a simulation of the situation without drift using similar physical parameters [14].…”

Section: Resultsmentioning

confidence: 99%

“…Secondly, we calibrated conditionally independent intervals of the time series with that particular length. We used the power-spectrum method as described previously [19] with the freely available program [20]. Finally, we calculated various types of variances.…”

Section: Methodsmentioning

confidence: 99%

Quantifying and pinpointing sources of noise in optical tweezers experiments

et al. 2009

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One limitation on the performance of optical traps is the noise inherently present in every setup. Therefore, it is the desire of most experimentalists to minimize and possibly eliminate noise from their optical trapping experiments. A step in this direction is to quantify the actual noise in the system and to evaluate how much each particular component contributes to the overall noise. For this purpose we present Allan variance analysis as a straightforward method. In particular, it allows for judging the impact of drift which gives rise to low-frequency noise, which is extremely difficult to pinpoint by other methods. We show how to determine the optimal sampling time for calibration, the optimal number of data points for a desired experiment, and we provide measurements of how much accuracy is gained by acquiring additional data points. Allan variances of both micrometersized spheres and asymmetric nanometer-sized rods are considered.

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tweezercalib 2.1: Faster version of MatLab package for precise calibration of optical tweezers

Cited by 41 publications

References 3 publications

Helical buckling of actin inside filopodia generates traction

Helical buckling of actin inside filopodia generates traction

Light at work: The use of optical forces for particle manipulation, sorting, and analysis

Quantifying and pinpointing sources of noise in optical tweezers experiments

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