J. Chloë Bulinski

Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells

Fluorescence microscopic visualization of fluorophore-conjugated proteins that have been microinjected or expressed in living cells and have incorporated into cellular structures has yielded much information about protein localization and dynamics [1]. This approach has, however, been limited by high background fluorescence and the difficulty of detecting movement of fluorescent structures because of uniform labeling. These problems have been partially alleviated by the use of more cumbersome methods such as three-dimensional confocal microscopy, laser photobleaching and photoactivation of fluorescence [2]. We report here a method called fluorescent speckle microscopy (FSM) that uses a very low concentration of fluorescent subunits, conventional wide-field fluorescence light microscopy and digital imaging with a low-noise, cooled charged coupled device (CCD) camera. A unique feature of this method is that it reveals the assembly dynamics, movement and turnover of protein assemblies throughout the image field of view at diffraction-limited resolution. We found that FSM also significantly reduces out-of-focus fluorescence and greatly improves visibility of fluorescently labeled structures and their dynamics in thick regions of living cells. Our initial applications include the measurement of microtubule movements in mitotic spindles and actin retrograde flow in migrating cells.

Visualization of microtubule growth in living platelets reveals a dynamic marginal band with multiple microtubules

The marginal band of microtubules maintains the discoid shape of resting blood platelets. Although studies of platelet microtubule coil structure conclude that it is composed of a single microtubule, no investigations of its dynamics exist. In contrast to previous studies, permeabilized platelets incubated with GTP-rhodamine-tubulin revealed tubulin incorporation at 7.9 (+/- 1.9) points throughout the coil, and anti-EB1 antibodies stained 8.7 (+/- 2.0) sites, indicative of multiple free microtubules. To pursue this result, we expressed the microtubule plus-end marker EB3-GFP in megakaryocytes and examined its behavior in living platelets released from these cells. Time-lapse microscopy of EB3-GFP in resting platelets revealed multiple assembly sites within the coil and a bidirectional pattern of assembly. Consistent with these findings, tyrosinated tubulin, a marker of newly assembled microtubules, localized to resting platelet microtubule coils. These results suggest that the resting platelet marginal band contains multiple highly dynamic microtubules of mixed polarity. Analysis of microtubule coil diameters in newly formed resting platelets indicates that microtubule coil shrinkage occurs with aging. In addition, activated EB3-GFP-expressing platelets exhibited a dramatic increase in polymerizing microtubules, which travel outward and into filopodia. Thus, the dynamic microtubules associated with the marginal band likely function during both resting and activated platelet states.

Accompanying protein alterations in malignant cells with a microtubule-polymerizing drug-resistance phenotype and a primary resistance mechanism

Microtubules (MTs) are cytoskeletal components whose structural integrity is mandatory for the execution of many basic cell functions. Utilizing parental and drug-resistant ovarian carcinoma cell lines that have acquired point mutations in beta-tubulin and p53, we studied the level of expression and modification of proteins involved in apoptosis and MT integrity. Extending previous results, we demonstrated phosphorylation of pro-survival Bcl-x(L) in an epothilone-A resistant cell line, correlating it with drug sensitivity to tubulin-active compounds. Furthermore, Mcl-1 protein turned over more rapidly following exposure to tubulin-modifying agents, the stability of Mcl-1 protein paralleling the drug sensitivity profile of the paclitaxel or epothilone-A resistant cell lines. The observed decreases in Mcl-1 were not a consequence of G(2)M arrest, as determined by flow cytometry analysis, which showed prominent levels of Mcl-1 in the absence of any drug treatment in populations enriched in mitotic cells. We also observed that a paclitaxel-resistant cell line expressed Bax at a much lower level than the sensitive parental line [A2780(1A9)], consistent with its mutant p53 status. MT-associated protein-4 (MAP4), whose phosphorylation during specific phases of the cell cycle reduces its MT-polymerizing and -stabilizing capabilities, was phosphorylated in response to drug challenge without a change in expression. Phosphorylation of MAP4 correlated with sensitivity to tubulin-binding drugs and with a dissociation from MTs. We propose that the tubulin mutations, which result in a compromised paclitaxel:tubulin or epothilone:tubulin interaction and paclitaxel or epothilone resistance, indirectly inhibit downstream events that lead to cell death, and this, in turn, may contribute to the drug-resistance phenotype

Mechanisms of trafficking in axons and dendrites: implications for development and neurodegeneration.

In the area of routing and sorting of dendritic traffic, the current phenomenological data beg questions about the cellular mechanisms utilized not only to transport material but also to modulate activity in a process, even apoptosis. To aid in formulating testable hypotheses, many plausible models are developed here and linked with some of the preliminary data that supports them. We first assume that in long dendrites the sorting of membranous proteins into transport vesicles also involves the linkage of motor proteins to the vesicles. Second, we assume that the cytoskeleton in dendrites is altered from the cytoskeleton in axons and the cell body. Viral glycoproteins, MAP2 and specific mRNA sorting into dendrites provide the simplest models for analyzing vesicular, cytoskeletal and RNA sorting. In the case of viral glycoproteins, initial sorting appears to occur at the Golgi but additional routing steps involve further complexities that could best be served by an additional sorting step at the junction of the cell body and the process. Transport of the specialized cytoskeletal proteins and specific mRNAs as well as vesicular material could be controlled by a similar gatekeeper at the mouth of a process. Studies of the microtubule-organelle motor complex, regulation of microtubule-based motility by microtubule-associated proteins, and slow axonal transport all provide insights into important aspects of the routing and sorting. These processes are in turn controlled by extracellular signals such as those generated by matrix molecules or their hydrolysis products in the case of amyloid precursor protein (APP). Routing and sorting mechanisms may be central to the development of Alzheimers disease in view of evidence that APP processing is affected, transport is disturbed, and intracellular vesicles (early endosomes) hypertrophied. Further it is possible that routing mechanisms play a role in cell-cell interactions as, for example, the possibility that pathogenic/cellular stress signals may be passed along circuits transsynaptically.

Microtubule Modification: Acetylation Speeds Anterograde Traffic Flow

Microtubules in neurites undergo multiple post-translational modifications. Recent work shows that neurites enriched in acetylated microtubules selectively support kinesin-mediated transport of the JNK regulator JIP-1 to growth cones.

A microplate assay for Leishmania amazonensis promastigotes expressing multimeric green fluorescent protein.

Abstract. Convenient and economical assays capable of screening many compounds are vital to advance the development of drug therapy. This is particularly important for many of the infections that occur mainly in the Third World. The development of such a spectrofluorometric assay for the protozoan parasite Leishmania is presented here. Using multimeric (four monomers) green fluorescent protein (GFP), Leishmania amazonensis promastigotes were generated with brightness measurable in 96-well microtiter plates. The promastigotes maintained the parental characteristics, were infective to murine macrophages and to mice, and the level of GFP fluorescence corresponded to the number of inoculated cells. The feasibility of using this assay for testing drugs kinetically and in a concentration-dependent manner, under microplate culture condition, was demonstrated with amphotericin B and the herbicide oryzalin, respectively. This assay is the first to allow a real-time analysis of antileishmanial agents with live promastigotes. The method of expressing multimeric GFP for in vitro drug screening is likely to be extendable to many species of parasitic protozoa.

CHANGES IN DENDRITIC STRUCTURE AND FUNCTION FOLLOWING HIPPOCAMPAL LESIONS : CORRELATIONS WITH DEVELOPMENTAL EVENTS?

Recovery after nervous system lesions may lead to partial re-institution of developmental schemes and processes. Here we review several of these proposed schemes, with the conclusion that though some processes may involve re-expression of embryonic phenotypes, there are many processes invoked during recovery from lesions that do not mirror developmental phenomena. The inability to fully revert to embryonic schemes because of adult phenotype may partially account for the decreased recovery observed in adults compared to that noted after lesions during development.

Growth factor priming differentially modulates components of the extracellular matrix proteome in chondrocytes and synovium-derived stem cells.

To make progress in cartilage repair it is essential to optimize protocols for two-dimensional cell expansion. Chondrocytes and SDSCs are promising cell sources for cartilage repair. We previously observed that priming with a specific growth factor cocktail (1 ng/mL transforming growth factor-β1, 5 ng/mL basic fibroblast growth factor, and 10 ng/mL platelet-derived growth factor-BB) in two-dimensional culture, led to significant improvement in mechanical and biochemical properties of synovium-derived stem cell (SDSC)-seeded constructs. The current study assessed the effect of growth factor priming on the proteome of canine chondrocytes and SDSCs. In particular, growth factor priming modulated the proteins associated with the extracellular matrix in two-dimensional cultures of chondrocytes and SDSCs, inducing a partial dedifferentiation of chondrocytes (most proteins associated with cartilage were down-regulated in primed chondrocytes) and a partial differentiation of SDSCs (some collagen-related proteins were up-regulated in primed SDSCs). However, when chondrocytes and SDSCs were grown in pellet culture, growth factor-primed cells maintained their chondrogenic potential with respect to glycosaminoglycan and collagen production. In conclusion, the strength of the label-free proteomics technique is that it allows for the determination of changes in components of the extracellular matrix proteome in chondrocytes and SDSCs in response to growth factor priming, which could help in future tissue engineering strategies.

Tubulin Posttranslational Modifications: A Pushmi-Pullyu at Work?

Numerous posttranslational modifications alter surface-exposed residues of tubulin within stable microtubules. The significance of one modification, glycylation, characteristic of ciliary and flagellar microtubules, has been particularly elusive. Two groups now identify the glycylation enzymes and determine the developmental consequences of their depletion. Glycylation enzymes and those responsible for another modification, glutamylation, work in opposition to one another in modifying microtubules.

Increased Stability of Microtubules in Cultured Olfactory Neuroepithelial Cells from Individuals with Schizophrenia.

Microtubules (MTs) are essential components of the cytoskeleton that play critical roles in neurodevelopment and adaptive central nervous system functioning. MTs are essential to growth cone advance and ultrastructural events integral to synaptic plasticity; these functions figure significantly into current pathophysiologic conceptualizations of schizophrenia. To date, no study has directly investigated MT dynamics in humans with schizophrenia. We therefore compared the stability of MTs in olfactory neuroepithelial (OE) cells between schizophrenia cases and matched nonpsychiatric comparison subjects. For this purpose, we applied nocodazole (Nz) to cultured OE cells obtained from tissue biopsies from seven living schizophrenia patients and seven matched comparison subjects; all schizophrenia cases were on antipsychotic medications. Nz allows MT depolymerization to be followed but prevents repolymerization, so that in living cells treated for varying time intervals, the MTs that are stable for a given treatment interval remain. Our readout of MT stability was the time at which fewer than 10 MTs per cell could be distinguished by anti-β-tubulin immunofluorescence. The percentage of cells with ≥10 intact MTs at specified intervals following Nz treatment was estimated by systematic uniform random sampling with Visiopharm software. These analyses showed that the mean percentages of OE cells with intact MTs were significantly greater for schizophrenia cases than for the matched comparison subjects at 10, 15, and 30min following Nz treatment indicating increased MT stability in OE cells from schizophrenia patients (p=0.0007 at 10min; p=0.0008 at 15min; p=0.036 at 30min). In conclusion, we have demonstrated increased MT stability in nearly all cultures of OE cells from individuals with schizophrenia, who received several antipsychotic treatments, versus comparison subjects matched for age and sex. While we cannot rule out a possible confounding effect of antipsychotic medications, these findings may reflect analogous neurobiological events in at least a subset of immature neurons or other cell types during gestation, or newly generated cells destined for the olfactory bulb or hippocampus, suggesting a mechanism that underlies findings of postmortem and neuroimaging investigations of schizophrenia. Future studies aimed at replicating these findings, including samples of medication-naïve subjects with schizophrenia, and reconciling the results with other studies, will be necessary. Although the observed abnormalities may suggest one of a number of putative pathophysiologic anomalies in schizophrenia, this work may ultimately have implications for an improved understanding of pathogenic processes related to this disorder.