An electro‐optic monitor of the behavior of Chlamydomonas reinhardtii cilia

Abstract

The unicellular green alga Chlamydomonas reinhardtii steers through water with a pair of cilia (eukaryotic flagella). Long-term observation of the beating of its cilia with controlled stimulation is improving our understanding of how a cell responds to sensory inputs. Here we describe how to record ciliary motion continuously for long periods. We also report experiments on the network of intracellular signaling that connects the environment inputs with response outputs. Local spatial changes in ciliary response on the time scale of the underlying biochemical dynamics are observed. Near-infrared light monitors the cells held by a micropipette. This condition is tolerated well for hours, not interfering with ciliary beating or sensory transduction. A computer integrates the light stimulation of the eye of Chlamydomonas with the ciliary motion making possible long-term correlations. Measures of ciliary responses include the beating frequency, stroke velocity, and stroke duration of each cilium, and the relative phase of the cis and trans cilia. The stationarity and dependence of the system on light intensity was investigated. About 150,000,000 total beat cycles and up to 8 h on one cell have been recorded. Each beat cycle is resolved so that each asynchronous beat is detected. Responses extend only a few hundred milliseconds, but there is a persistence of momentary changes that last much longer. Interestingly, we see a response that is linear with absolute light intensity as well as different kinds of response that are clearly nonlinear, implying two signaling pathways from the cell body to the cilia.

The DPY-30 Domain and Its Flanking Sequence Mediate the Assembly and Modulation of Flagellar Radial Spoke Complexes

“…As flagellum-propelled movements are mercurial and remain poorly understood (29,47), we tentatively offer several speculations regarding the asynchrony and steering anomaly of RSP2 cells. This reminded us of the spinning tendency of a transgenic mutant defective in a calcium-binding motif of an outer dynein docking protein (9).…”

“…The spokehead contacts the CP intermittently, and the contact is crucial for flagellar beating and synchrony (62,66). It has been shown that bright light selectively enhances the inherent asynchrony of only one (trans) flagellum of an algal cell (29,47). Perhaps RSP2's C-terminal tail and calmodulin strengthen the spokehead, constitutively or in a calcium-dependent manner, to regulate the asynchrony rate.…”

Ciliary behavior of a negatively phototactic Chlamydomonas reinhardtii

“…Three-dimensional motion can be inferred by slight adjustments in focus necessary during visual observation to bring different parts of each cilium into focus, also seen in previous work [Rüffer and Nultsch, 1991]. The relative phase changes to square-wave stimulation occurring between the anterior and posterior quadrants of a single cilium [Josef et al, 2005] suggests either a nonplanar motion or a change in the orientation at the base of the cilium that is proportional to absolute light intensity. The detector signal amplitude and, therefore, velocity recorded will reflect changes in the ciliary beat plane.…”

Linear systems analysis of the ciliary steering behavior associated with negative‐phototaxis in Chlamydomonas reinhardtii

“…Signals from sensory transduction control gene expression, initiate internal biochemical signals, change the membrane potential, and perturb the cell away from its ionic homeostasis. Analysis of a number of measures of the ciliary beating response suggests that ciliary beating is controlled by multiple processes occurring in parallel [Josef et al, 2005a]. There are many light regulable inputs that can be concurrently modulated and multiple outputs that can be observed dynamically in real time as the cell makes decisions based on the regulated and internal inputs.…”

“…2). The rhodopsin photoreceptors are stimulated with modulated green light (543 nm) and the movements of each cilium are recorded [Josef et al, 2005a]. Chlamydomonas phototaxis responses are quite time-invariant over the ciliary monitoring interval ($2-5 h), provided the system is adapted (conditioned) prior to recording.…”