Gianni Piperno

Primary cilia of human endothelial cells disassemble under laminar shear stress

We identified primary cilia and centrosomes in cultured human umbilical vein endothelial cells (HUVEC) by antibodies to acetyl-α-tubulin and capillary morphogenesis gene-1 product (CMG-1), a human homologue of the intraflagellar transport (IFT) protein IFT-71 in Chlamydomonas. CMG-1 was present in particles along primary cilia of HUVEC at interphase and around the oldest basal body/centriole at interphase and mitosis. To study the response of primary cilia and centrosomes to mechanical stimuli, we exposed cultured HUVEC to laminar shear stress (LSS). Under LSS, all primary cilia disassembled, and centrosomes were deprived of CMG-1. We conclude that the exposure to LSS ends the IFT in cultured endothelial cells.

[23] Detection of acetylated α-tubulin by specific antibodies

Publisher Summary The molecules of α- and β-tubulin found in microtubules may be posttranslationally modified in various ways. One such modification is the acetylation of the side chain of lysine residues of α-tubulin. The acetylation may be a step in a pathway leading to microtubule stabilization. This reaction is reversible and occurs within 1 min after microtubule assembly. It could be fast enough to regulate in part the rapid transitions of the microtubules between growing and depolymerizing phases. This chapter presents immunological methods used to study this modification. Although the procedures were designed to study α-tubulin acetylation per se, they also have been put to use in a different direction: they allow a very sensitive staining of neurons in histological preparations. 6-11 B-1 antibody has been used to detect α-tubulins acetylated on Lys-40 from a variety of organisms. Other antibodies specific for acetylated a-tubulin have been described: the other six described by Piperno and Fuller all share the 6-11 B-1 binding site.

Two Flagellar Genes, AGG2 and AGG3, Mediate Orientation to Light in Chlamydomonas

Ciliary membranes have a large repertoire of receptors and ion channels that act to transduce information from the environment to the cell. Chlamydomonas offers a tractable system for dissecting the transport and function of ciliary and flagellar membrane proteins. Isolation of ergosterol and sphingolipid-enriched Chlamydomonas flagellar membrane domains identified potential signaling molecules by mass spectroscopy. These include a membrane protein and a matrix flavodoxin protein that are encoded by the AGG2 and AGG3 genes, respectively. Agg2p localizes to the proximal flagellar membrane near the basal bodies. Agg3p is distributed throughout the flagellar matrix, with an increased concentration in the proximal regions where Agg2p is located. Chlamydomonas cells sense light by using a microbial-type rhodopsin , transduce a signal from the cell body to the flagella, and alter the waveform of the flagella to turn a cell toward the light. Protein depletion by RNA interference reveals that both AGG gene products play roles in the orientation of cells to a directional light source. The depleted strains mimic the phenotype of the previously identified agg1 mutant, which swims away from light. We propose that the localization of Agg2p and Agg3p to the proximal region of the flagella may be important for interpreting light signals.

ISOLATION OF RADIAL SPOKE HEADS FROM CHLAMYDOMONAS AXONEMES

Publisher Summary There are three binding sites in the radial spoke heads of Chlamydomonas axonemes. They are bound to the central microtubule complex, paired by a thin fiber, and connected through a stalk to the A-tubule of the outer-doublet microtubules. The interactions occurring at these binding sites result in a modification of axonemal waveforms that is necessary for efficient swimming and displacement of the cell body. The radial spoke heads of Chlamydomonas are considered as a model system for studying organelle assembly. They can be partially purified as a protein complex and are composed of six distinct proteins. Five components of the radial spoke head complex are missing from flagella of distinct Chlamydomonas mutants. These mutants are paralyzed and have radial spoke components as defective gene products. Rescue of radial spoke heads in axonemes of dikaryons between a radial spoke mutant and a wild-type strain requires coassembly of radial spoke proteins from both strains. The procedure described in this chapter was developed for partially purifying radial spoke heads from Chlamydomonas axonemes. It should be applicable to the isolation of radial spoke heads from axonemes of other systems, assuming that in these systems radial spoke head components form a complex and can be separated from radial spoke stalks as in Chlamydomonas axonemes. The procedure for isolation of radial spoke heads from Chlamydomonas axonemes are based on the preferential solubilization of radial spoke heads. That solubilization occurs under conditions of low ionic strength. The axonemes are previously extracted with 0.5 M NaCl and 1 m M ATP-Mg to remove the outer and inner dynein arms. The final step of the procedure consists of a sedimentation of solubilized radial spoke heads in a sucrose gradient. This step does not separate the radial spoke heads from the tubulin subunits. Column chromatography would be necessary.

TWO-DIMENSIONAL SEPARATION OF AXONEMAL PROTEINS

Publisher Summary The procedure described in this chapter is meant to resolve the molecular components of Chlamydomonas axonemes. It can be applied to all kinds of axonemes for various purposes—namely, the analysis of axonemal fractions, the identification of defective gene products, the recognition of cytoskeletal proteins, and, finally, the definition of “molecular signatures” that are formed by specific substructures. These signatures consist of characteristic two-dimensional patterns of spots. The development of two-dimensional electrophoretic procedures capable of resolving nearly all axonemal proteins provided the opportunity to identify the molecular components of all major axonemal substructures. Axoneme is composed of hundreds of distinct polypeptides that have different concentrations and are post-translationally modified. The procedure discussed consists of two successive gel electrophoreses of polypeptides that, first, are resolved in a pH gradient and, then, are separated by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). During the first step the polypeptides retain their own electric charge but do not reach their isoelectric point. The procedure works very well for separating proteins that are smaller than 200,000 Da. Larger proteins need analysis by one-dimensional SDS-PAGE. The electrophoreses are performed in two homemade, vertical apparatus that hold 16-cm-wide, 1Pcm-long, 0.3-cm-thick glass plates and 35.5-cm-wide, 26.5-cm-long, 0.4-cm-thick glass plates, respectively. The dimensions of the apparatus for the second electrophoresis are designed so that two samples can be run side-by-side in an identical acrylamide gradient. The procedure is described in detail. Following the second electrophoresis, detection of polypeptides is performed either by silver staining of wet gels or autoradiography of dried gels. In nonequilibrium electrophoresis of polypeptides in a gradient of pH, all polypeptides under analysis remain in the gel. Polypeptides that could irreversibly precipitate at their isoelectric point in a focusing gel remain soluble and can be separated in the gel used for the second electrophoresis.

Electrophoretic separation of dynein heavy chains.

Publisher Summary Molecular studies of axonemal dyneins have demonstrated that each outer and inner dynein arm contains at least two multiple dynein heavy chains (DHCs) and that inner dynein arms may be composed of different heavy chains along the length of the axoneme. It is probable that each kind of motile axoneme contains multiple DHCs. All DHCs characterized until now cannot be resolved by two-dimensional poly-acrylamide gel electrophoresis and are resolved by one-dimensional polyacrylamide gel electrophoresis only under specific conditions. The electrophoretic procedure presented in this chapter is a modified version of the procedure of Neville. It can be applied to resolve at least some of the DHCs present in cilia or eukaryotic flagella. It was developed to obtain optimal resolution of the 9 or 11 DHCs that are assembled in the axoneme of Chlamydomonas flagella. The procedure is capable of resolving six bands, each comprising one or two or three of the DHCs. Although changes in the composition of the gel or buffers used for the electrophoresis cause changes in the electrophoretic patterns formed by the DHCs, all DHCs cannot be separated by gel electrophoresis alone because they are so similar in structure and present in the axonemes at different concentrations. Complete separation of DHC subsets can be achieved through gel electrophoresis if the number of DHCs under analysis is reduced by mutation or chromatographic fractionation. The chapter discusses the chemicals involved, the solutions, and the apparatus needed. Nine stock solutions are prepared and kept at room temperature. The electrophoresis is performed in a homemade, vertical apparatus that holds 35.5-cm-wide, 26.5-cm-long, and 0.4-cm-thick glass plates. Thinner glass plates are not adequate for optimal resolution of dynein heavy chains because they are flexible and do not provide a gel slab that has homogeneous thickness. Polymerizations are performed at room temperatures.