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Companion Web Site for The Chlamydomonas SourcebookThe documents and videos presented here are supplements to volumes 2 and 3 of The Chlamydomonas Sourcebook, Second Edition.
Supplementary Material for Volume 2Fluorescence Quenching AnalysisThis document, which supplements Chapter 14 by Jun Minagawa, describes a versatile technique to diagnose light-harvesting functions.
Methods for Analysis of Photosystem II in ChlamydomonasThis document is a supplement to Chapter 16, by Zoee Gokhale and Richard T. Sayre. It contains the following sections: Preparation of Thylakoid Membranes
Supplementary Material for Volume 3: Chlamydomonas VideosMotility and behavior of course mean movement, and many of the processes discussed in Volume 3 are best represented by movies. Over the years, some spectacular movies of Chlamydomonas have been made. Many of these movies are referred to in Volume 3, and we are pleased to provide them on this website. Some of the movies found here have not previously been widely available, and some are provided in a longer version than has been available previously. We hope that you take some time to view these movies, because they show, with a piquancy that words alone cannot convey, why so many of us continue to be excited and enthralled by Chlamydomonas.
Bacteria moving on flagella [Bloodgood]A non-motile mutant of the bacterium E. coli (strain M-247) is used as a marker to visualize force production at the flagellar surface of Chlamydomonas reinhardtii, paralyzed strain pf18. The bacterium adheres to the flagellar membrane and is being moved in a bidirectional, saltatory manner.
Cell moving along bead [Bloodgood]Polystyrene microspheres (0.36 µm in diameter) are adherent to the glass slide as well as suspended in the buffer solution. A live Chlamydomonas reinhardtii cell of the paralyzed strain pf18 has adhered to an immobilized group of microspheres by its flagellar surface. In applying force to the microsphere in an attempt to move it, the cell is actually translocating itself bidirectionally relative to the immobilized microsphere (at the same time that other microspheres are moving freely along the flagellar surface). The velocity of translocation of the whole cell is the same as that of individual microspheres moving along the flagellar surface despite the enormous difference in load in the two cases.
Central pair rotating in Chlamydomonas axoneme [Kamiya]
This video shows rotation of a partially extruded central pair extending from the end of a demembranated flagellum in a reactivated Chlamydomonas cell model. Dark-field images were taken with a silicon-intensified target camera. Because a mirror was inserted between the ocular and camera, the image is a mirror image. The cell body is rotated by the frictional force between the rotating central pair and the surface of the glass slide. (See Kamiya, R. 1982. Extrusion and rotation of the central-pair microtubules in detergent-treated Chlamydomonas flagella. Cell Motil. Suppl. 1, 169-173.)
Central pair rotating in demembranated Chlamydomonas cell model [Kamiya]This movie shows the central pair of microtubules rotating and extruding from a demembranated flagellar axoneme that has detached from a Chlamydomonascell. Note that the central pair takes on a corkscrew shape as it is extruded. Dark-field microscopy, images recorded using a silicon-intensified target camera. (See Kamiya, R. 1982. Extrusion and rotation of the central-pair microtubules in detergent-treated Chlamydomonasflagella. Cell Motil. Suppl. 1, 169-173.)
Chlamydomonas for dinner? [Pickett-Heaps]This video with sound, provided courtesy of Jeremy and Julianne Pickett-Heaps and Cytographics (Cytographics.com), records the heliozoan protist Actinophrys in the act of capturing its prey, in this case Chlamydomonas. The heliozoan has a spherical cell body with thick radiating strands of cytoplasm called axopodia, which are supported by microtubules. When smaller organisms bump into the axopodia, they stick to the axopodia and are then moved down the axopodia to the cell body, where they are phagocytosed. Chlamydomonas adheres to the axopodia by its flagella but not its cell body, and has evolved the ability to escape by shedding its flagella (shown in the last sequence). As a result, the heliozoan has flagella for dinner, while the Chlamydomonas cell floats away and lives to grow new flagella.
Chlamy_IFT_Kozminski [Kozminski et al.]One low-magnification sequence, repeated twice, followed by one high-magnification sequence, illustrating intraflagellar transport (IFT) in Chlamydomonas moewusii, non-swimming strain M475. IFT is the movement of large protein complexes, called IFT particles, from the base to the tip of the flagellum (anterograde movement), and then back to the base of the flagellum (retrograde movement). Particles are moving at ~2.0 µm/s in the anterograde direction and at ~3.5 µm/s in the retrograde direction. The cell body is out of focus except for its very apex, which appears as a bright spot. Video-enhanced differential-interference contrast microscopy, real time. The high-magnification sequence is also shown in the accompanying video "Chlamy_IFT_closeup_Kozminski."
Chlamy_IFT_closeup_Kozminski [Kozminski et al.]This high magnification view of a single flagellum attached to a Chlamydomonas moewusii cell of the non-swimming strain M475 shows intraflagellar transport (IFT) - the rapid bidirectional traffic of small particles within the flagellum. These particles, which are large protein complexes (and NOT membrane-bounded organelles), are moving at ~2.0 µm/second in the base-to-tip (anterograde) direction and at ~3.5 µm/second in the tip-to-base (retrograde) direction. IFT is essential for the assembly and maintenance of almost all cilia and flagella. Video-enhanced differential-interference contrast microscopy, real time.
Chlamy photoshock response [Hegemann lab]Movement and photophobic responses of Chlamydomonas reinhardtii recorded in real time. Chlamydomonas cells (2x105 cells per ml) were placed in a 0.4-ml fluorescence cuvette under the microscope (Zeiss Axiovert 100; 5x objective). The light path through the cuvette was 2 mm. Movement was recorded at 25 frames per second under infrared-light (RG8 filter, Schott) using a dark-field condensor and a commercial/conventional DVD recorder. Actinic flashes of green light (510 nm; 10 microsecond) were applied through the objective (epi illumination) to induce the photophobic response.
Chlamy phototaxis and photoshock [Witman lab]This video shows Chlamydomonas cells exhibiting strong negative phototaxis, and near the end, several photoshock events. The recording is in real time. The cells are imaged by dark-field microscopy using red light (625-nm cutoff), to which the cells are relatively insensitive. Near the top of the field of view is a time record indicating hours, minutes, and seconds, respectively. Initially, the cells are unstimulated and are swimming in random directions. At ~00:00:21 on the time record, the digits representing hours go out, indicating that a blue (488 nm) stimulus beam, to which the cells respond strongly, has been introduced from the left. Because the cells are negatively phototactic, they begin swimming to the right, away from the light source. At ~ 00:00:38, the digits representing hours come back on, and those representing minutes go off, indicating that the stimulus beam has been switched to the opposite side of the chamber so that light is now coming from the right; the cells immediately turn and begin swimming to the left. At ~00:01:00, the stimulus beam is turned off, and cells gradually return to swimming in random directions. This is followed by a series of bright flashes to illustrate the photoshock response.
Electron Microscopic Tomogram Videos [O'Toole and Dutcher] Cross section tomogram of distal region of wild type basal bodies Longitudinal tomogram of wild type basal bodies Proximal and distal regions joined
FMG1 Antibody Movement [Bloodgood]Live Chlamydomonas reinhardtii were exposed to a monoclonal antibody to the FMG-1B major flagellar membrane glycoprotein. This antibody crosslinked and induced a large cluster of the FMG-1B glycoproteins to be translocated bidirectionally within the plane of the flagellar membrane. The cluster of glycoproteins exhibits saltatory movement at a velocity similar to the velocity of microsphere movements and whole-cell gliding motility.
Gliding [Kozminski et al.] Two cells of Chlamydomonas moewusii, strain M475 (a non-swimming mutant), exhibit whole-cell gliding motility, which is a bi-directional form of cell motility dependent upon close contact of the flagellar surface with a solid substrate. Careful inspection reveals that intraflagellar transport (IFT) is occurring within each of the flagella on these cells; IFT is the bidirectional transport of protein complexes called IFT particles. Video-enhanced differential-interference contrast microscopy, real time.
Gliding and Microsphere [Kozminski et al.]This Chlamydomonas moewusii cell of the non-swimming strain M475 is exhibiting three active forms of motility. The cell is gliding along the solid substrate, utilizing the contact of the flagellar surface with the glass slide. There are polystyrene microspheres (0.3 µm in diameter) moving along the surface of each of the two flagella. Note that the direction of microsphere motility is not linked to the direction of gliding motility. Within each flagellum, there is intraflagellar transport (IFT) -- the active trafficking of small particles. Video-enhanced differential-interference contrast microscopy, real time.
IFT27-GFP video [Qin et al.]Chlamydomonas IFT27-GFP undergoes intraflagellar transport (IFT). IFT27-GFP transgenic cells were paralyzed with 20 mM NaPPi to facilitate recording. IFT27-GFP movement in the flagella was recorded with a Nikon Eclipse TE2000 microscope. Images were collected at 100 ms per frame. The movie was created with Metamorph software. The movie displayed is played in real time. (From Qin et al., 2007. Intraflagellar transport protein 27 is a small G protein involved in cell-cycle control. Curr. Biol. 17, 193-202.).
KAP-GFP [Mueller et al.]Real-time imaging of a GFP-tagged KAP subunit in Chlamydomonas reinhardtii. The fla3 mutant was transformed with a wild-type copy of the KAP gene containing a C-terminal GFP tag.
Microsphere 1 [Bloodgood]A single polystyrene microsphere (0.36-µm diameter) exhibits bidirectional, saltatory movement along the surface of a flagellum of Chlamydomonas reinhardtii, paralyzed strain pf18. The microsphere can pause and reverse direction at any point along the flagellar surface.
Microsphere 2 [Bloodgood]A number of polystyrene microspheres (0.36 µm in diameter) are adhering to and being translocated along the surfaces of the two flagella of this Chlamydomonas reinhardtii cell of the paralyzed strain pf18. Each microsphere is exhibiting movement independent from that of the other microspheres on the same flagellum. Microspheres moving in opposite directions can be seen to pass each other, showing that there must be local force transduction involving small domains of the flagellar membrane (probably representing local cross-linked patches of the FMG-1B flagellar membrane glycoprotein).
MicrosphereMove [Kozminski et al.]This movie shows the bidirectional saltatory movement of 0.3-µm diameter polystyrene microspheres along the surface of a flagellum of a Chlamydomonas moewusii cell of the non-swimming strain M475. Video-enhanced differential-interference contrast microscopy, real time. Elsevier's DisclaimerInformation provided in this document is provided "as is" without warranty of any kind, either express or implied. Every effort has been made to ensureaccuracy and conformance to standards accepted at the time of publication. The reader is advised to research other sources of information on these topics. The user assumes the entire risk as to the accuracy and the use of this document. This document may be copied and distributed subject to the following conditions: 1. All text must be copied without modification and all pages must be included; |
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