Cutting off ciliary protein import:intraflagellar transport after dendritic femtosecond-laser ablation

Primary cilia, organelles protruding from the surface of eukaryotic cells, act as cellular antennae to detect and transmit signals from the extracellular environment. They are built and maintained by continuous cycles of intraflagellar transport (IFT), where ciliary proteins are transported between the ciliary base and tip. These proteins originate from the cell body because cilia lack protein synthesis machinery. How input from the cell body affects IFT and ciliary function is not well understood. Here, we use femtosecond-laser ablation to perturb the dendritic input of proteins to chemosensory cilia in living Caenorhabditis elegans. Using fluorescence microscopy, we visualize and quantify the real-time response of ciliary proteins to dendritic ablation. We find that the response occurs in three distinct stages. First, IFT dynein is activated within seconds, redistributing IFT components toward the ciliary base; second, the ciliary axoneme shortens and motors slow down; and third, motors leave the cilium. Depletion of ATP by adding azide also results in IFT slowdown and IFT components leaving the cilium, but not in activation of retrograde IFT. These results indicate that laser ablation triggers a specific mechanism important for IFT regulation that allows the cilium to rapidly adapt to changes in the outside environment

Extending the bandwidth of optical-tweezers interferometry

The extension of the bandwidth of optical-tweezers interferometry was discussed. It was found that the detection bandwidth was extended to at least 100 KHz, either by using wavelengths below 850 nm or by using different detectors at longer wavelengths. The power spectral density of the Brownian motion of micron-sized beads in optical tweezers was also measured

Power spectrum analysis for optical tweezers. II: Laser wavelength dependence of parasitic filtering, and how to achieve high bandwidth

In a typical optical tweezers detection system, the position of a trapped object is determined from laser light impinging on a quadrant photodiode. When the laser is infrared and the photodiode is of silicon, they can act together as an unintended low-pass filter. This parasicit effect is due to the high transparency of silicon to near-infrared light. A simple model that accounts for this phenomenon is here solved for frequencies up to 100 kHz and for laser wavelengths between 750 and 1064 nm. The solution is applied to experimental data in the same range, and is demonstrated to give this detection system of optical tweezers a bandwidth, accuracy, and precision that are limited only by the data acquisition board's bandwidth and bandpass ripples, here 96.7 kHz and 0.005 dB, respectively. ©2006 American Institute of Physic

Why motor proteins team up - Intraflagellar transport in C. elegans cilia

Inside the cell, vital processes such as cell division and intracellular transport are driven by the concerted action of different molecular motor proteins. In C. elegans chemosensory cilia, 2 kinesin-2 family motor proteins, kinesin-II and OSM-3, team up to drive intraflagellar transport (IFT) in the anterograde direction, from base to tip, whereas IFT dynein hitchhikes toward the tip and subsequently drives IFT in the opposite, retrograde direction, thereby recycling both kinesins. While it is evident that at least a retrograde and an anterograde motor are necessary to drive IFT, it has remained puzzling why 2 same-polarity kinesins are employed. Recently, we addressed this question by combining advanced genome-engineering tools with ultrasensitive, quantitative fluorescence microscopy to study IFT with single-molecule sensitivity.(1,2) Using this combination of approaches, we uncovered a differentiation in kinesin-2 function, in which the slower kinesin-II operates as an 'importer', loading IFT trains into the cilium before gradually handing them over to the faster OSM-3. OSM-3 subsequently acts as a long-range 'transporter', driving the IFT trains toward the tip. The two kinesin-2 motors combine their unique motility properties to achieve something neither motor can achieve on its own; that is to optimize the amount of cargo inside the cilium. In this commentary, we provide detailed insight into the rationale behind our research approach and comment on our recent findings. Moreover, we discuss the role of IFT dynein and provide an outlook on future studies

Laser-induced heating in optical traps

In an optical tweezers experiment intense laser light is tightly focused to intensities of MW/cm(2) in order to apply forces to submicron particles or to measure mechanical properties of macromolecules. It is important to quantify potentially harmful or misleading heating effects due to the high light intensities in biophysical experiments. We present a model that incorporates the geometry of the experiment in a physically correct manner, including heat generation by light absorption in the neighborhood of the focus, balanced by outward heat flow, and heat sinking by the glass surfaces of the sample chamber. This is in contrast to the earlier simple models assuming heat generation in the trapped particle only. We find that in the most common experimental circumstances, using micron-sized polystyrene or silica beads, absorption of the laser light in the solvent around the trapped particle, not in the particle itself, is the most important contribution to heating. To validate our model we measured the spectrum of the Brownian motion of trapped beads in water and in glycerol as a function of the trapping laser intensity. Heating both increases the thermal motion of the bead and decreases the viscosity of the medium. We measured that the temperature in the focus increased by 34.2 +/- 0.1 K/W with 1064-nm laser light for 2200-nm-diameter polystyrene beads in glycerol, 43.8 +/- 2.2 K/W for 840-nm polystyrene beads in glycerol, 41.1 +/- 0.7 K/W for 502-nm polystyrene beads in glycerol, and 7.7 +/- 1.2 K/W for 500-nm silica beads and 8.1 +/- 2.1 K/W for 444-nm silica beads in water. Furthermore, we observed that in glycerol the heating effect increased when the bead was trapped further away from the cover glass/glycerol interface as predicted by the model. We show that even though the heating effect in water is rather small it can have non-negligible effects on trap calibration in typical biophysical experimental circumstances and should be taken into consideration when laser powers of more than 100 mW are used

Replication Data for: IFT cargo and motors associate sequentially with IFT trains to enter cilia

The dataset contains the data and the MATLAB scripts associated with the manuscript titled IFT cargo and motors associate sequentially with IFT trains to enter cilia Here we provide scripts and data corresponding to four different aspects: (1) single-molecule tracking data for all the IFT-components studied in the paper (2) numerical simulations of single-molecule entry events to estimate real pause time. (3) numerical simuations for obtaining 1D projection information from simulated 2D localization data along a hollow cylinder (4) Spline fitting to transform global xy-coordinates of single-molecule tracks to spline coordinates The data and codes are orgainized in separate folders. Each folder contains a ReadMe.txt file which details the data (and structure) and the accompanying script(s). Data is stored as .csv (or xlsx) files and scripts are written in MATLAB (The Math Works, Inc., R2021a)