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36 records · Page 2Linked to original sources
PubMed2004

A polarity dependent fluorescence "switch" in live cells.

The spectroscopic properties, ultrafast kinetics and utilization of a photochromic molecule as a bi-stable fluorescing sensor of polarity in live cells are described. This molecule is a photochromic fulgimide, 2,3-dialkylidenesuccinimide, which emits fluorescence that can be switched optically on and off. The fluorescence intensity is a function of the polarity of the molecular environment, namely it fluoresces strongly when the molecule is in its polar isomeric structure form. We demonstrate that this molecule enters live cells without inducing damage, it binds primarily to internal membranous organelles (mitochondria) and its fluorescence can be switched optically "on" and "off" repeatedly while inside the living cell. A possible use as a bi-stable, on/off sensor is discussed.

Animals↗
PubMed2006

Lysed cell models and isolated chromosomes for the study of kinetochore/centromere biochemistry in vitro.

The centromere or kinetochore functions in both chromosome movement and in regulation of progression through mitosis. It appears likely that the signaling pathways involved are keenly dependent on solid phase cytoskeletal and karyoskeletal scaffolds that may mediate important physical signals such as tension. Understanding these pathways will be greatly aided by reconstructing the signaling in lysed cell models. Here we present approaches to the in vitro study of signaling pathways in mitotic cells, particularly those involved in protein phosphorylation changes at kinetochores that may control cell cycle progression in M phase.

Animals↗
PubMed2006

Defining the regulatory elements in the proximal promoter of DeltaNp63 in keratinocytes: Potential roles for Sp1/Sp3, NF-Y, and p63.

p63, a homolog of the tumor suppressor p53, plays an important role in the formation of stratified epithelium such as those in the epidermis of the skin. The p63 gene gives rise to multiple functionally distinct protein isoforms, including the DeltaNp63 class of isoforms, which lacks the N-terminal transactivation domain and is synthesized from an internal promoter. DeltaNp63 proteins are the predominant isoforms expressed in keratinocytes and are thought to be important for maintenance of the proliferative capacity of these cells. Here, we have examined the transcriptional control mechanisms that govern the expression DeltaNp63 in keratinocytes. We first performed DNase I hypersensitive site mapping and demonstrated that the promoter region of DeltaNp63 is in open chromatin state in keratinocytes. To identify the cis-elements that regulate DeltaNp63, we have performed transient transfection assays in keratinocytes with several DeltaNp63 promoter constructs. This identified a short evolutionarily conserved fragment that harbors most of the transcriptional activity of the DeltaNp63 promoter. Biochemical studies of this element have revealed critical roles for CCAAT-box-binding factor (CBF/NF-Y) and Sp1/Sp3 family of proteins. In addition, our data suggest that DeltaNp63 is recruited to and can activate its own promoter, possibly through protein-protein interactions, thus providing an auto-regulatory loop of self-regulation. These studies support the notion that unique and distinct pathways control the expression of individual p53 family members and their various isoforms.

Animals↗
PubMed2005

Taxol-stabilized microtubules can position the cytokinetic furrow in mammalian cells.

How microtubules act to position the plane of cell division during cytokinesis is a topic of much debate. Recently, we showed that a subpopulation of stable microtubules extends past chromosomes and interacts with the cell cortex at the site of furrowing, suggesting that these stabilized microtubules may stimulate contractility. To test the hypothesis that stable microtubules can position furrows, we used taxol to rapidly suppress microtubule dynamics during various stages of mitosis in PtK1 cells. Cells with stabilized prometaphase or metaphase microtubule arrays were able to initiate furrowing when induced into anaphase by inhibition of the spindle checkpoint. In these cells, few microtubules contacted the cortex. Furrows formed later than usual, were often aberrant, and did not progress to completion. Images showed that furrowing correlated with the presence of one or a few stable spindle microtubule plus ends at the cortex. Actin, myosin II, and anillin were all concentrated in these furrows, demonstrating that components of the contractile ring can be localized by stable microtubules. Inner centromere protein (INCENP) was not found in these ingressions, confirming that INCENP is dispensable for furrow positioning. Taxol-stabilization of the numerous microtubule-cortex interactions after anaphase onset delayed furrow initiation but did not perturb furrow positioning. We conclude that taxol-stabilized microtubules can act to position the furrow and that loss of microtubule dynamics delays the timing of furrow onset and prevents completion. We discuss our findings relative to models for cleavage stimulation.

Anaphase↗
PubMed2004

Signal analysis of total internal reflection fluorescent speckle microscopy (TIR-FSM) and wide-field epi-fluorescence FSM of the actin cytoskeleton and focal adhesions in living cells.

Fluorescent speckle microscopy (FSM) uses low levels of fluorescent proteins to create fluorescent speckles on cytoskeletal polymers in high-resolution fluorescence images of living cells. The dynamics of speckles over time encode subunit turnover and motion of the cytoskeletal polymers. We sought to improve on current FSM technology by first expanding it to study the dynamics of a non-polymeric macromolecular assembly, using focal adhesions as a test case, and second, to exploit for FSM the high contrast afforded by total internal reflection fluorescence microscopy (TIR-FM). Here, we first demonstrate that low levels of expression of a green fluorescent protein (GFP) conjugate of the focal adhesion protein, vinculin, results in clusters of fluorescent vinculin speckles on the ventral cell surface, which by immunofluorescence labelling of total vinculin correspond to sparse labelling of dense focal adhesion structures. This demonstrates that the FSM principle can be applied to study focal adhesions. We then use both GFP-vinculin expression and microinjected fluorescently labelled purified actin to compare quantitatively the speckle signal in FSM images of focal adhesions and the actin cytoskeleton in living cells by TIR-FM and wide-field epifluorescence microscopy. We use quantitative FSM image analysis software to define two new parameters for analysing FSM signal features that we can extract automatically: speckle modulation and speckle detectability. Our analysis shows that TIR-FSM affords major improvements in these parameters compared with wide-field epifluorescence FSM. Finally, we find that use of a crippled eukaryotic expression promoter for driving low-level GFP-fusion protein expression is a useful tool for FSM imaging. When used in time-lapse mode, TIR-FSM of actin and GFP-conjugated focal adhesion proteins will allow quantification of molecular dynamics within interesting macromolecular assemblies at the ventral surface of living cells.

Actins↗
PubMed2005

In vivo selective cytoskeleton dynamics quantification in interphase cells induced by pulsed ultraviolet laser nanosurgery.

We report on the manipulation of intracellular filaments using a nanosurgery system based on a subnanosecond pulsed UV laser optimized for the localized severing of biological polymers. By inducing artificial catastrophe of selected microtubules (MTs), we perform shrinkage-rate measurements in interphase Ptk-2 cells throughout the entire cell. We quantify the impact of two labeling methods and three fluorescent markers, showing a 25% faster depolymerization with Alexa-488 tubulin compared with Rhodamine and yellow fluorescent protein (YFP) tubulins and a 20% higher variability induced by microinjection compared with stable transfection. Using EB3-GFP as a tip marker, we establish a new protocol to measure shrinkage rate, growth rate and rescue frequency simultaneously with high temporal and spatial specificity in live cells. As our analysis shows, laser-induced MT dynamics are physiologically relevant. The high statistical efficiency that the method offers in terms of numbers of measured events and therefore reduced standard deviations represents an important quantitative improvement in the measurement of dynamic instability parameters in vivo. We extend the application of the method by demonstrating induced dynamic behavior of actin-stress fibers after severing. This new method enables the quantitative investigation of cytoskeleton dynamics in a local confinement.

Animals↗
PubMed2006

Chromosomes can congress to the metaphase plate before biorientation.

The stable propagation of genetic material during cell division depends on the congression of chromosomes to the spindle equator before the cell initiates anaphase. It is generally assumed that congression requires that chromosomes are connected to the opposite poles of the bipolar spindle ("bioriented"). In mammalian cells, we found that chromosomes can congress before becoming bioriented. By combining the use of reversible chemical inhibitors, live-cell light microscopy, and correlative electron microscopy, we found that monooriented chromosomes could glide toward the spindle equator alongside kinetochore fibers attached to other already bioriented chromosomes. This congression mechanism depended on the kinetochore-associated, plus end-directed microtubule motor CENP-E (kinesin-7).

Animals↗
PubMed2007

Differential transmission of actin motion within focal adhesions.

Cell migration requires the transmission of motion generated in the actin cytoskeleton to the extracellular environment through a complex assembly of proteins in focal adhesions. We developed correlational fluorescent speckle microscopy to measure the coupling of focal-adhesion proteins to actin filaments. Different classes of focal-adhesion structural and regulatory molecules exhibited varying degrees of correlated motions with actin filaments, indicating hierarchical transmission of actin motion through focal adhesions. Interactions between vinculin, talin, and actin filaments appear to constitute a slippage interface between the cytoskeleton and integrins, generating a molecular clutch that is regulated during the morphodynamic transitions of cell migration.

Actin Cytoskeleton↗
PubMed2006

Deciphering protein function during mitosis in PtK cells using RNAi.

BACKGROUND: Studying mitosis requires a system in which the dramatic movements of chromosomes and spindle microtubules can be visualized. PtK cells, due to their flat morphology and their small number of large chromosomes, allow microscopic visualizations to be readily performed. RESULTS: By performing RNAi in PtK cells, we can explore the function of many proteins important for spindle assembly and chromosome segregation. Although it is difficult to transfect DNA into PtK cells (efficiency approximately 10%), we have transfected a fluorescent siRNA at nearly 100% efficiency. Using a cDNA expression library, we then isolated a complete PtK MCAK (P-MCAK) cDNA. P-MCAK shares 81% identity to Human-MCAK (H-MCAK) protein and 66% identity to H-MCAK DNA. Knockdown of P-MCAK by RNAi caused defects in chromosome congression and defective spindle organization. Live imaging revealed that chromosomes had defects in congression and segregation, similar to what we found after microinjection of inhibitory anti-MCAK antibodies. Because it is laborious to isolate full-length clones, we explored using RT-PCR with degenerate primers to yield cDNA fragments from PtK cells from which to design siRNAs. We isolated a cDNA fragment of the mitotic kinesin Eg5 from PtK cells. This fragment is 93% identical to H-Eg5 protein and 87% identical to H-Eg5 DNA. A conserved 21 bp siRNA was used for RNAi in both HeLa and PtK cells in which Eg5 knockdown resulted in an increased mitotic index and cells with monopolar spindles. In addition, we used RT-PCR to isolate fragments of 5 additional genes, whose sequence identity ranged from 76 to 90% with human, mouse, or rat genes, suggesting that this strategy is feasible to apply to any gene of interest. CONCLUSION: This approach will allow us to effectively probe mitotic defects from protein knockdowns by combining genomic information from other organisms with the tractable morphology of PtK cells.

Amino Acid Sequence↗
PubMed2006

Decreased polarity and increased random motility in PtK1 epithelial cells correlate with inhibition of endosomal recycling.

Locomoting cells exhibit a constant retrograde flow of plasma membrane proteins from the leading edge towards the cell center, which, when coupled to substrate adhesion, may drive forward cell movement. Here, we aimed to test the hypothesis that, in epithelial cells, these plasma membrane components are delivered via a polarized endo/exocytotic cycle, and that their correct recycling is required for normal migration. To this end, we expressed in PtK1 cells cDNA constructs encoding GDP-restricted (S25N) and GTP-restricted (Q70L) mutants of Rab11b, a small GTPase that has been implicated in the late stage of recycling, where membrane components from the endosomal recycling compartment are transported back to the plasma membrane. Surprisingly, we found that transient expression of the Rab11b mutants in randomly migrating PtK1 cells in small cell islands caused altered cell morphology and actually increased the velocity of cell locomotion. Stable expression of either mutant protein also did not decrease cell migration velocity, but instead affected the directionality of migration in monolayer wound healing assays. We have also tested the effects of other Rab proteins, implicated in endocytic recycling, and discovered a clear correlation between the degree of recycling inhibition and the increase in non-directional cell motility.

Animals↗
PubMed2005

Periodic patterns of actin turnover in lamellipodia and lamellae of migrating epithelial cells analyzed by quantitative Fluorescent Speckle Microscopy.

We measured actin turnover in lamellipodia and lamellae of migrating cells, using quantitative Fluorescent Speckle Microscopy. Lamellae disassembled at low rates from the front to the back. However, the dominant feature in their turnover was a spatially random pattern of periodic polymerization and depolymerization moving with the retrograde flow. Power spectra contained frequencies between 0.5 and 1 cycle/min. The spectra remained unchanged when applying Latrunculin A and Jasplakinolide in low doses, except that additional frequencies occurred beyond 1 cycle/min. Whereas Latrunculin did not change the rate of mean disassembly, Jasplakinolide halted it completely, indicating that the steady state and the dynamics of actin turnover are differentially affected by pharmacological agents. Lamellipodia assembled in recurring bursts at the leading edge and disassembled approximately 2.5 microm behind. Events of polymerization correlated spatially and temporally with transient formation of Arp2/3 clusters. In lamellae, Arp2/3 accumulation and polymerization correlated only spatially, suggesting an Arp2/3-independent mechanism for filament nucleation. To acquire these data we had to enhance the resolution of quantitative Fluorescent Speckle Microscopy to the submicron level. Several algorithmic advances in speckle image processing are described enabling the analysis of kinetic and kinematic activities of polymer networks at the level of single speckles.

Actin-Related Protein 2↗
PubMed2005

Bacterial shape and ActA distribution affect initiation of Listeria monocytogenes actin-based motility.

We have examined the process by which the intracellular bacterial pathogen Listeria monocytogenes initiates actin-based motility and determined the contribution of the variable surface distribution of the ActA protein to initiation and steady-state movement. To directly correlate ActA distributions to actin dynamics and motility of live bacteria, ActA was fused to a monomeric red fluorescent protein (mRFP1). Actin comet tail formation and steady-state bacterial movement rates both depended on ActA distribution, which in turn was tightly coupled to the bacterial cell cycle. Motility initiation was found to be a highly complex, multistep process for bacteria, in contrast to the simple symmetry breaking previously observed for ActA-coated spherical beads. F-actin initially accumulated along the sides of the bacterium and then slowly migrated to the bacterial pole expressing the highest density of ActA as a tail formed. Early movement was highly unstable with extreme changes in speed and frequent stops. Over time, saltatory motility and sensitivity to the immediate environment decreased as bacterial movement became robust at a constant steady-state speed.

Actins↗
PubMed2005

Lamellipodial contractions during crawling and spreading.

Most eukaryotic cells can crawl over surfaces. In general, this motility requires three distinct actions: polymerization at the leading edge, adhesion to the substrate, and retraction at the rear. Recent experiments with mouse embryonic fibroblasts showed that during spreading and crawling the lamellipodium undergoes periodic contractions that are substrate-dependent. Here I show that a simple model incorporating stick-slip adhesion and a simplified mechanism for the generation of contractile forces is sufficient to explain periodic lamellipodial contractions. This model also explains why treatment of cells with latrunculin modifies the period of these contractions. In addition, by coupling a diffusing chemical species that can bind actin, such as myosin light-chain kinase, with the contractile model leads to periodic rows and waves in the chemical species, similar to what is observed in experiments. This model provides a novel and simple explanation for the generation of contractile waves during cell spreading and crawling that is only dependent on stick-slip adhesion and the generation of contractile force and suggests new experiments to test this mechanism.

Actins↗
PubMed2006

In vivo quantitative studies of dynamic intracellular processes using fluorescence correlation spectroscopy.

It has been a significant challenge to quantitatively study the dynamic intracellular processes in live cells. These studies are essential for a thorough understanding of the underlying mechanisms regulating the signaling pathways and the transitions between cell cycle stages. Our studies of Cdc20, an important mitotic checkpoint protein, throughout the cell cycle demonstrate that fluorescence correlation spectroscopy is a powerful tool for in vivo quantitative studies of dynamic intracellular processes. In this study, Cdc20 is found to be present primarily in a large complex (>1 Mda) during interphase with a diffusion constant of 1.8+/-0.1 microm2/s and a concentration of 76+/-24 nM, consistent with its association with the APC/C. During mitosis, however, a proportion of Cdc20 dissociates from APC/C at a rate of 12 pM/s into a soluble pool with a diffusion constant of 19.5+/-5.0 microm2/s, whose size is most consistent with free Cdc20. This free pool accumulates to 50% of total Cdc20 (approximately 40 nM) during chronic activation of the mitotic checkpoint but disappears during mitotic exit at a rate of 31 pM/s. The observed changes in the biochemical assembly states of Cdc20 closely correlate to the known temporal pattern of the activity of APC/CCdc20 in mitosis. Photon counting histograms reveal that both complexes contain only a single molecule of Cdc20. The underlying mechanisms of the activities of APC/CCdc20 throughout the cell cycle are discussed in light of our experimental observations.

Cdc20 Proteins↗