Near real-time measurement of forces applied by an optical trap to a rigid cylindrical object

2016

PLoS ONE

Self Cite

Although solitary or sensory cilia are present in most cells of the body and their existence has been known since the sixties, very little is known about their functions. One suspected function is fluid flow sensing- physical bending of cilia produces an influx of Ca++, which can then result in a variety of activated signaling pathways. Defective cilia and ciliary-associated proteins have been shown to result in cystic diseases. Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a progressive disease, typically appearing in the 5th decade of life and is one of the most common monogenetic inherited human diseases, affecting approximately 600,000 people in the United States. Because the mechanical properties of cilia impact their response to applied flow, we asked how the stiffness of cilia can be controlled pharmacologically. We performed an experiment subjecting cilia to Taxol (a microtubule stabilizer) and CoCl2 (a HIF stabilizer to model hypoxia). Madin-Darby Canine Kidney (MDCK) cells were selected as our model system. After incubation with a selected pharmacological agent, cilia were optically trapped and the bending modulus measured. We found that HIF stabilization significantly weakens cilia. These results illustrate a method to alter the mechanical properties of primary cilia and potentially alter the flow sensing properties of cilia.

“…The spatial dynamics of the diffracted beam were recorded using a quadrant photodiode (QPD) and the data analyzed as per [47]. …”

Section: Methodsmentioning

confidence: 99%

“…However, because our data processing algorithm [47], while insensitive to the shape of a trapped object, was checked only for free objects, the fact that trapped cilia are anchored at one end could potentially invalidate our data processing method. We checked this by applying the trap to different locations along the ciliary axoneme.…”

Section: Sources Of Errormentioning

confidence: 99%

HIF Stabilization Weakens Primary Cilia

2016

PLoS ONE

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“…Our experimental protocols for the growth, maintainance, and pharmacological manipulations of ciliated epithelial cells and optical trapping of cilia have been published elsewhere (15,20,22), so here we only provide a brief summary. Conditionally immortalized epithelial cells originally microdissected from the cortical collecting duct of an Immortomouse are grown to confluence and allowed to differentiate for several days, during which time a cilium emerges.…”

Section: Methodsmentioning

confidence: 99%

“…The primary sources of independent random error are: calculation of the MSD asymptote, measurement of cilium length, and experiment-to-experiment variability of the trap stiffness. As discussed in (22), the variability of MSD asymptote is reduced due to processing independent data subsets, the variation of this parameter is typically 10%. The variation due to length measurement varies with length because our uncertainty is fixed by the depth of focus of the objective lens, δL = ±0.3µm.…”

Section: Sources Of Random Errormentioning

confidence: 99%

Mechanical Properties of a Primary Cilium from the Stochastic Motions of the Cilium Tip

Feng

Peng

et al. 2018

Preprint

The stochastic tip dynamics of a primary cilium held within an optical trap is quantified by combining experimental, analytical and computational tools. Primary cilia are cellular organelles, present on most vertebrate cells, hypothesized to function as a fluid flow sensor. The mechanical properties of a cilium remain incompletely characterized. We measured the fluctuating position of an optically trapped cilium tip under untreated, Taxol-treated, and HIF-stabilized conditions. We applied analytical modeling to derive the mean-squared displacement of the trapped tip of a cilium and compared the results with experimental measurements. We provide, for the first time, evidence that the effective flexural rigidity of a ciliary axoneme is length-dependent, and longer cilia are stiffer than shorter cilia. We then provide a rational explanation for both effects. We demonstrate that the apparent length-dependent flexural rigidity can be understood by a combination of modeling axonemal microtubules orthotropic elastic shells and including (actin-driven) active stochastic basal body motion. It is hoped that our improved characterization of cilia will result in deeper understanding of the biological function of cellular flow sensing by this organelle. Our model could be profitably applied to motile cilia and our results also demonstrate the possibility of using easily observable ciliary dynamics to probe interior cytoskeletal dynamics. Experimental RationaleHere, we consider the primary cilium as a passive mechanical beam capable of transducing kinetic energy of flowing fluid into elastic strain (bending) energy. Bending the cilium seems to be required to initiate calcium signaling (2), and the amount of bending in response to applied flow can be characterized by the flexural rigidity (equivalently, bending modulus) (14) 'EI'. Even so, the mechanical response of primary cilia to external loading remains incompletely characterized (15). Most prior measurements of the ciliary bending modulus fall within the range of 10-50 pN*µm 2 (14), (16), but there are also measurements that report significantly higher bending modulus, as high as 300 pN*µm 2 (17). As we will show and discuss, our results potentially resolve this discrepancy but also raise important new questions. It is essential to note that prior to this report, all reported measurements of the ciliary bending modulus, with the excepton of (15), calculate the bending modulus based on fitting an image of a bent cilium to the equation for a homogeneous cantilevered beam (discussed below, in 'Analysis'). Our approach is qualitatively different and does not rely on imaging the cilium.Measuring the strained position of the cilium tip provides information about the mechanical response of the cilium to an applied mechanical load. For example, subjecting a uniform cantilever of length 'L' to a static load 'q' localized to the free end results in a static tip displacement y = q L 3 /3EI. When both L and y are known, the expression can be inverted to calculate an 'effective' bendin...

“…The spatial dynamics of the diffracted beam were recorded using a quadrant photodiode (QPD) and the data analyzed as per Glaser et al (52). Briefly, the QPD outputs the centroid location of the diffracted trapping beam, digitally sampled at 10 kS/s.…”

Section: Optical Tweezersmentioning

confidence: 99%

Mechanical Properties of a Primary Cilium As Measured by Resonant Oscillation

2015

Biophysical Journal

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Primary cilia are ubiquitous mammalian cellular substructures implicated in an ever-increasing number of regulatory pathways. The well-established ciliary hypothesis states that physical bending of the cilium (for example, due to fluid flow) initiates signaling cascades, yet the mechanical properties of the cilium remain incompletely measured, resulting in confusion regarding the biological significance of flow-induced ciliary mechanotransduction. In this work we measure the mechanical properties of a primary cilium by using an optical trap to induce resonant oscillation of the structure. Our data indicate 1) the primary cilium is not a simple cantilevered beam; 2) the base of the cilium may be modeled as a nonlinear rotatory spring, with the linear spring constant k of the cilium base calculated to be (4.6 ± 0.62) × 10(-12) N/rad and nonlinear spring constant α to be (-1 ± 0.34) × 10(-10) N/rad(2); and 3) the ciliary base may be an essential regulator of mechanotransduction signaling. Our method is also particularly suited to measure mechanical properties of nodal cilia, stereocilia, and motile cilia-anatomically similar structures with very different physiological functions.