Roger D. Sloboda

The Microtubule Plus End-Tracking Protein EB1 Is Localized to the Flagellar Tip and Basal Bodies in Chlamydomonas reinhardtii

Flagellar axonemes assemble and continuously turn over at the flagellar tip. The supply and removal of axonemal subunits at the tip are mediated by intraflagellar transport (IFT), a motility process essential for the assembly and maintenance of all eukaryotic flagella and cilia. IFT is characterized by the movement of large protein complexes (IFT particles) from the basal bodies to the flagellar tip by kinesin-II and from the tip back to the basal bodies by cytoplasmic dynein 1b. The IFT particles consist of approximately 16 polypeptides partitioned into two complexes, A and B, and associate with axonemal precursors/turn over products. The mechanisms by which IFT motor regulation and cargo loading/unloading occur at the flagellar tip are unknown. We identified a Chlamydomonas reinhardtii ortholog of the microtubule (MT) plus end-tracking protein EB1 [4] (CrEB1) and show here that CrEB1 localizes to the tip of flagella and to the proximal part of the basal bodies. Furthermore, we found that CrEB1 is depleted from flagella of the temperature-sensitive (ts) flagellar assembly-defective (fla) mutant fla11(ts) at the restrictive temperature. This depletion of CrEB1 is accompanied by a dramatic accumulation of IFT particle polypeptides near the flagellar tip.

[39] Purification and assay of microtubule-associated proteins (MAPs)

Publisher Summary This chapter presents procedure for purification and assay of microtubule-associated proteins (MAPS). The procedures begin with microtubules assembled in vitro from either brain or tissue culture cells. The solution of microtubule proteins is usually carried through at least two to three complete assembly-disassembly cycles prior to beginning one of the following MAP isolation procedures. The strategies employed to isolate MAPs from the total microtubule protein preparation are based on several criteria: (1) net charge distribution affecting their behavior on ion exchange resins, (2) overall molecular size and axial ratio, which affect their elution from molecular sieve columns, and (3) varying stability to the effects of elevated temperatures (100°). One or more combinations of these properties can be used as the basis for the isolation procedures. Both the high molecular weight MAPs and Tau from brain and the MAPs from tissue culture cells have been assayed by their ability to promote the assembly of tubulin in vitro. Generally, these assays are carried out by determining whether the putative MAP will enhance the initial rate and total amount of assembly of a given concentration of PC or DEAE-purified brain tubulin.

Autofluorescence characteristics of immortalized and carcinogen-transformed human bronchial epithelial cells

Tissue autofluorescence has been explored as a potential method of noninvasive pre-neoplasia (pre-malignancy) detection in the lung. Here, we report the first studies of intrinsic cellular autofluorescence from SV40 immortalized and distinct tobacco-carcinogen-transformed (malignant) human bronchial epithelial cells. These cell lines are useful models for studies seeking to distinguish between normal and pre-neoplastic human bronchial epithelial cells. The cells were characterized via spectrofluorimetry and confocal fluorescence microscopy. Spectrofluorimetry revealed that tryptophan was the dominant fluorophore. No change in tryptophan emission intensity was observed between immortalized and carcinogen-transformed cells. Confocal autofluorescence microscopy was performed using a highly sensitive, spectrometer-coupled instrument capable of limiting emission detection to specific wavelength ranges. These studies revealed two additional endogenous fluorophores, whose excitation and emission characteristics were consistent with nicotinamide adenine dinucleotide (NADH) and flavins. In immortalized human bronchial epithelial cells, the fluorescence of these species was localized to cytoplasmic granules. In contrast, the carcinogen-transformed cells showed an appreciable decrease in the fluorescence intensity of both NADH and flavins and the punctate, spatial localization of the autofluorescence was lost. The observed autofluorescence decrease was potentially the result of changes in the redox state of the fluorophores. The random cytoplasmic fluorescence pattern found in carcinogen-transformed cells may be attributed to changes in the mitochondrial morphology. The implications of these results to pre-neoplasia detection in the lung are discussed.

Calcium and calmodulin-dependent phosphorylation of a 62 kd protein induces microtubule depolymerization in sea urchin mitotic apparatuses

Sea urchin mitotic apparatuses (MAs) were isolated in a microtubule stabilizing buffer that contained detergent. These isolated MAs contain a calcium and calmodulin-dependent protein kinase that phosphorylates one specific MA-associated endogenous substrate with a relative molecular mass of 62 kd. No protein phosphorylation occurs in the presence of calcium or magnesium ion alone, or when magnesium ion is combined with 10 microM cyclic AMP or cyclic GMP. Because in vivo labeling studies showed that the 62 kd protein was also phosphorylated in living cells during mitosis, the effect of protein phosphorylation on MA stability was also studied. When isolated MAs were incubated under conditions that resulted in phosphorylation of the 62 kd protein, substantial depolymerization of MA microtubules occurred within 10 min. MAs incubated under similar conditions but in the absence of 62 kd phosphorylation lost many fewer microtubules and were stable for up to 30 min. The results are discussed with respect to a model for mitosis in which the specific role of protein phosphorylation in the events of anaphase is addressed.

In Vivo NADH fluorescence monitoring as an assay for cellular damage in photodynamic therapy

Abstract In this study the endogenous fluorescence signal attributed to reduced nicotinamide adenine dinucleotide (NADH) has been measured in response to photodynamic therapy (PDT)–induced damage. Measurements on cells in vitro have shown that NADH fluorescence decreased relative to that of controls after treatment with a toxic dose of PDT, as measured within 30 min after treatment. Similarly, assays of cell viability indicated that mitochondrial function was reduced immediately after treatment in proportion to the dose delivered, and the proportion of this dose response did not degrade further over 24 h. Measurements in vivo were used to monitor the fluorescence emission spectrum and the excited state lifetime of NADH in PDT-treated tissue. The NADH signal was defined as the ratio of the integrated fluorescence intensity of the 450 ± 25 nm emission band relative to the fluorescence intensity integrated over the entire 400–600 nm range of collection. Measurements in murine muscle tissue indicated a 22% reduction in the fluorescence signal immediately after treatment with verteporfin-based PDT, using a dose of 2 mg/kg injected 15 min before a 48 J/cm2 light dose at 690 nm. Control animals without photosensitizer injection had no significant change in the fluorescence signal from laser irradiation at the same doses. This signal was monotonically correlated to the deposited dose used here and could provide a direct dosimetric measure of PDT-induced cellular death in the tissue being treated.

Intraflagellar transport and the flagellar tip complex.

Intraflagellar transport (IFT) is the term that refers to the microtubule dependent particle motility that is common to almost all flagella and cilia and is distinct from the mechanism of flagellar beating. IFT involves the rapid, bi‐directional transport of molecular motors and their cargo proteins from the base to the tip of the flagellum and back again. While the basic mechanism of IFT is well established, the varied functions of this process are continually being elucidated. For example, although IFT plays a clear role in flagellar assembly, disassembly and stability, the exact sequence of events that take place when tubulin subunit addition and loss occur during flagellar assembly and disassembly, respectively, are unknown. Key to furthering our understanding of IFT is greater knowledge of the flagellar tip complex (FTC) because it is at the FTC that flagellar assembly and disassembly, cargo loading and unloading, and motor protein regulation occur. Yet these related processes may only represent one aspect of the importance of IFT in flagellar dynamics. IFT may also provide the basic elements of a signal transduction mechanism that functions to provide the nucleus with information about the outside environment and even about the state of the flagellum itself. Thus, IFT may function as the central component of a signal transduction system that controls flagellar gene transcription.

Making sense of cilia and flagella

Data reported at an international meeting on the sensory and motile functions of cilia, including the primary cilium found on most cells in the human body, have thrust this organelle to the forefront of studies on the cell biology of human disease.

Microinjection of antibodies to a 62 kd mitotic apparatus protein arrests mitosis in dividing sea urchin embryos

Previously, we described a 62 kd protein that is a component of the isolated sea urchin mitotic apparatus. This protein is a substrate for an endogenous, calcium/calmodulin-dependent protein kinase also associated with the mitotic apparatus. Phosphorylation of the 62 kd protein directly correlates with the depolymerization of microtubules in isolated mitotic apparatuses. Here we report a test of the function of the 62 kd protein in vivo. Double labeling studies using a monoclonal antibody to tubulin and an affinity purified antibody specific for the 62 kd protein reveal that the 62 kd protein co-localizes with mitotic apparatus microtubules. When affinity purified antibodies to the 62 kd protein were microinjected into dividing sea urchin embryos, mitosis was blocked in a stage-specific manner. The results are discussed with respect to the role of the 62 kd protein in the metaphase-anaphase transition.

Griseofulvin: Association with tubulin and inhibition of in vitro microtubule assembly☆

Abstract Griseofulvin—shown previously to disrupt the mitotic apparatus in vivo —inhibited the in vitro microtubule assembly reaction completely at 8 × 10−4M griseofulvin. In a gel filtration assay, randomly tritiated griseofulvin associated stoichiometrically with purified tubulin, as determined by chromatography on Sephadex G-25. No detectable drug binding was observed when bovine serum albumin was used as a control in an identical column assay. Both gel filtration chromatography and a kinetic analysis of the inhibition of assembly by griseofulvin suggest that the drug interacts directly and stoichimetrically with the tubulin dimer, and that the interaction is both rapid and independent of temperature.

A Protein Methylation Pathway in Chlamydomonas Flagella Is Active during Flagellar Resorption

During intraflagellar transport (IFT), the regulation of motor proteins, the loading and unloading of cargo and the turnover of flagellar proteins all occur at the flagellar tip. To begin an analysis of the protein composition of the flagellar tip, we used difference gel electrophoresis to compare long versus short (i.e., regenerating) flagella. The concentration of tip proteins should be higher relative to that of tubulin (which is constant per unit length of the flagellum) in short compared with long flagella. One protein we have identified is the cobalamin-independent form of methionine synthase (MetE). Antibodies to MetE label flagella in a punctate pattern reminiscent of IFT particle staining, and immunoblot analysis reveals that the amount of MetE in flagella is low in full-length flagella, increased in regenerating flagella, and highest in resorbing flagella. Four methylated proteins have been identified in resorbing flagella, using antibodies specific for asymmetrically dimethylated arginine residues. These proteins are found almost exclusively in the axonemal fraction, and the methylated forms of these proteins are essentially absent in full-length and regenerating flagella. Because most cells resorb cilia/flagella before cell division, these data indicate a link between flagellar protein methylation and progression through the cell cycle.