scholarly journals Microtubule dynamics in fish melanophores.

1994 ◽  
Vol 126 (6) ◽  
pp. 1455-1464 ◽  
Author(s):  
V I Rodionov ◽  
S S Lim ◽  
V I Gelfand ◽  
G G Borisy
Keyword(s):  
Intracellular Transport ◽  
Time Course ◽  
Dynamic Properties ◽  
Time Lapse ◽  
Microtubule Dynamics ◽  
Charge Coupled Device ◽  
Alpha Tubulin ◽  
Acetylated Tubulin ◽  
Microtubule Stability ◽  
Pigment Distribution

We have studied the dynamics of microtubules in black tetra (Gymnocorymbus ternetzi) melanophores to test the possible correlation of microtubule stability and intracellular particle transport. X-rhodamine-or caged fluorescein-conjugated tubulin were microinjected and visualized by fluorescence digital imaging using a cooled charge coupled device and videomicroscopy. Microtubule dynamics were evaluated by determining the time course of tubulin incorporation after pulse injection, by time lapse observation, and by quantitation of fluorescence redistribution after photobleaching and photoactivation. The time course experiments showed that the kinetics of incorporation of labeled tubulin into microtubules were similar for cells with aggregated or dispersed pigment with most microtubules becoming fully labeled within 15-20 min after injection. Quantitation by fluorescence redistribution after photobleaching and photoactivation confirmed that microtubule turnover was rapid in both states, t1/2 = 3.5 +/- 1.5 and 6.1 +/- 3.0 min for cells with aggregated and dispersed pigment, respectively. In addition, immunostaining with antibodies specific to posttranslationally modified alpha-tubulin, which is usually enriched in stable microtubules, showed that microtubules composed exclusively of detyrosinated tubulin were absent and microtubules containing acetylated tubulin were sparse. We conclude that the microtubules of melanophores are very dynamic, that their dynamic properties do not depend critically on the state of pigment distribution, and that their stabilization is not a prerequisite for intracellular transport.

scholarly journals Discrete regions of the kinesin-8 Kip3 tail differentially mediate astral microtubule stability and spindle disassembly

2018 ◽  
Vol 29 (15) ◽  
pp. 1866-1877 ◽  
Author(s):  
Sandeep Dave ◽  
Samuel J. Anderson ◽  
Pallavi Sinha Roy ◽  
Emmanuel T. Nsamba ◽  
Angela R. Bunning ◽  
...  
Keyword(s):  
Dynamic Properties ◽  
Microtubule Dynamics ◽  
Motor Domain ◽  
Spindle Positioning ◽  
Cellular Processes ◽  
Microtubule Stability ◽  
Astral Microtubule ◽  
Cellular Context ◽  
Spindle Disassembly ◽  
The Stability

To function in diverse cellular processes, the dynamic properties of microtubules must be tightly regulated. Cellular microtubules are influenced by a multitude of regulatory proteins, but how their activities are spatiotemporally coordinated within the cell, or on specific microtubules, remains mostly obscure. The conserved kinesin-8 motor proteins are important microtubule regulators, and family members from diverse species combine directed motility with the ability to modify microtubule dynamics. Yet how kinesin-8 activities are appropriately deployed in the cellular context is largely unknown. Here we reveal the importance of the nonmotor tail in differentially controlling the physiological functions of the budding yeast kinesin-8, Kip3. We demonstrate that the tailless Kip3 motor domain adequately governs microtubule dynamics at the bud tip to allow spindle positioning in early mitosis. Notably, discrete regions of the tail mediate specific functions of Kip3 on astral and spindle microtubules. The region proximal to the motor domain operates to spatially regulate astral microtubule stability, while the distal tail serves a previously unrecognized role to control the timing of mitotic spindle disassembly. These findings provide insights into how nonmotor tail domains differentially control kinesin functions in cells and the mechanisms that spatiotemporally control the stability of cellular microtubules.

scholarly journals Deep Multi-OMICs and Multi-Tissue Characterization in a Pre- and Postprandial State in Human Volunteers: The GEMM Family Study Research Design

Genes ◽  
2018 ◽  
Vol 9 (11) ◽  
pp. 532 ◽  
Author(s):  
Raul A. Bastarrachea ◽  
Hugo A. Laviada-Molina ◽  
Edna J. Nava-Gonzalez ◽  
Irene Leal-Berumen ◽  
Claudia Escudero-Lourdes ◽  
...  
Keyword(s):  
Research Design ◽  
Time Course ◽  
Tissue Characterization ◽  
Metabolic Diseases ◽  
Family Study ◽  
Dynamic Properties ◽  
Data Generation ◽  
Proteomic Profiling ◽  
Cvd Risk ◽  
Postprandial Metabolism

Cardiovascular disease (CVD) and type 2 diabetes (T2D) are increasing worldwide. This is mainly due to an unhealthy nutrition, implying that variation in CVD risk may be due to variation in the capacity to manage a nutritional load. We examined the genomic basis of postprandial metabolism. Our main purpose was to introduce the GEMM Family Study (Genetics of Metabolic Diseases in Mexico) as a multi-center study carrying out an ongoing recruitment of healthy urban adults. Each participant received a mixed meal challenge and provided a 5-hours’ time course series of blood, buffy coat specimens for DNA isolation, and adipose tissue (ADT)/skeletal muscle (SKM) biopsies at fasting and 3 h after the meal. A comprehensive profiling, including metabolomic signatures in blood and transcriptomic and proteomic profiling in SKM and ADT, was performed to describe tendencies for variation in postprandial response. Our data generation methods showed preliminary trends indicating that by characterizing the dynamic properties of biomarkers with metabolic activity and analyzing multi-OMICS data it could be possible, with this methodology and research design, to identify early trends for molecular biology systems and genes involved in the fasted and fed states.

scholarly journals Peroxisomes move by hitchhiking on early endosomes using the novel linker protein PxdA

2015 ◽  
Author(s):  
John Salogiannis ◽  
Martin J. Egan ◽  
Samara L. Reck-Peterson
Keyword(s):  
Molecular Motors ◽  
Intracellular Transport ◽  
Coiled Coil ◽  
Leading Edge ◽  
Time Lapse ◽  
Early Endosome ◽  
Organelle Transport ◽  
The Novel ◽  
Coiled Coil Region ◽  
Early Endosomes

Eukaryotic cells use microtubule-based intracellular transport for the delivery of many subcellular cargos, including organelles. The canonical view of organelle transport is that organelles directly recruit molecular motors via cargo-specific adaptors. In contrast to this view, we show here that peroxisomes move by hitchhiking on early endosomes, an organelle that directly recruits the transport machinery. Using the filamentous fungus Aspergillus nidulans we find that hitchhiking is mediated by a novel endosome-associated linker protein, PxdA. PxdA is required for normal distribution and long-range movement of peroxisomes, but not early endosomes or nuclei. Using simultaneous time-lapse imaging we find that early endosome-associated PxdA localizes to the leading edge of moving peroxisomes. We identify a coiled-coil region within PxdA that is necessary and sufficient for early endosome localization and peroxisome distribution and motility. These results present a new mechanism of microtubule-based organelle transport where peroxisomes hitchhike on early endosomes and identify PxdA as the novel linker protein required for this coupling.

scholarly journals Hierarchies of Host Factor Dynamics at the Entry Site of Shigella flexneri during Host Cell Invasion

2012 ◽  
Vol 80 (7) ◽  
pp. 2548-2557 ◽  
Author(s):  
Soudeh Ehsani ◽  
José Carlos Santos ◽  
Cristina D. Rodrigues ◽  
Ricardo Henriques ◽  
Laurent Audry ◽  
...  
Keyword(s):  
Host Cell ◽  
Tyrosine Kinases ◽  
Time Course ◽  
Shigella Flexneri ◽  
Time Lapse ◽  
Small Gtpases ◽  
Host Factors ◽  
Host Cells ◽  
Entry Site ◽  
Content Type

ABSTRACTShigella flexneri, the causative agent of bacillary dysentery, induces massive cytoskeletal rearrangement, resulting in its entry into nonphagocytic epithelial cells. The bacterium-engulfing membrane ruffles are formed by polymerizing actin, a process activated through injected bacterial effectors that target host small GTPases and tyrosine kinases. Once inside the host cell,S. flexneriescapes from the endocytic vacuole within minutes to move intra- and intercellularly. We quantified the fluorescence signals from fluorescently tagged host factors that are recruited to the site of pathogen entry and vacuolar escape. Quantitative time lapse fluorescence imaging revealed simultaneous recruitment of polymerizing actin, small GTPases of the Rho family, and tyrosine kinases. In contrast, we found that actin surrounding the vacuole containing bacteria dispersed first from the disassembling membranes, whereas other host factors remained colocalized with the membrane remnants. Furthermore, we found that the disassembly of the membrane remnants took place rapidly, within minutes after bacterial release into the cytoplasm. Superresolution visualization of galectin 3 through photoactivated localization microscopy characterized these remnants as small, specular, patchy structures between 30 and 300 nm in diameter. Using our experimental setup to track the time course of infection, we identified theS. flexnerieffector IpgB1 as an accelerator of the infection pace, specifically targeting the entry step, but not vacuolar progression or escape. Together, our studies show that bacterial entry into host cells follows precise kinetics and that this time course can be targeted by the pathogen.

The Marginal Microtubule Coil in the Resting Blood Platelet Is a Dynamic Bipolar Array.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1653-1653 ◽  
Author(s):  
Joseph E. Italiano ◽  
Jennifer L. Richardson ◽  
Harald Schulze ◽  
Ksenija Drabek ◽  
Chloe Bulinski ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Immunofluorescence Microscopy ◽  
Blood Platelet ◽  
Time Lapse ◽  
Microtubule Dynamics ◽  
Cell Periphery ◽  
Structural Studies ◽  
Time Lapse Microscopy ◽  
Marginal Band ◽  
Tyrosinated Tubulin

Abstract The discoid shape of the resting blood platelet is maintained by its marginal microtubule band. Structural studies have concluded that this band is composed of a single microtubule coiled 8-12 times around the cell periphery. To understand the dynamics of the microtubule coil, we took advantage of EB1 and EB3, proteins that highlight the ends of growing microtubules. Immunofluorescence microscopy with anti-EB1 revealed clear staining of numerous (8.7 +/− 2.0, range 4–12) comet-like dashes in the microtubule coil, suggesting the presence of several microtubule plus ends. Consistent with this observation, rhodamine-tubulin added to permeabilized platelets incorporates at multiple (7.9 +/−1.9) points throughout the microtubule coil. To visualize microtubule dynamics in platelets, we retrovirally directed megakaryocytes to express the microtubule plus-end marker EB3-GFP and isolated platelets released in these cultures. Fluorescence time-lapse microscopy of EB3-GFP-expressing resting platelets revealed multiple microtubule plus ends that grew in both clockwise and counterclockwise directions. Antibodies that recognize tyrosinated tubulin, which preferentially label newly assembled microtubules and not stable microtubules, stain the microtubule coil. These results indicate that resting platelets contain a bipolar array of microtubules that undergoes continuous assembly. When EB3-GFP-expressing platelets are activated with thrombin, the number of polymerizing microtubules increases dramatically and the microtubules grow into filopodia. Collectively, these results suggest that the marginal band of the resting blood platelet is highly dynamic, bipolar, and contains multiple microtubule plus ends. These ends are amplified in platelet activation and point towards the active edges of the cells and the tips of filopodia.

Tubacin, An Inhibitor of HDAC6, Induces Apoptosis of Acute Lymphoblastic Leukemia Cells in Vitro and in Vivo through a Na+/K+ATPase-Dependent Pathway.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1923-1923 ◽  
Author(s):  
Agustin Rodriguez-Gonzalez ◽  
Tiffany Simms-Waldrip ◽  
Alan K. Ikeda ◽  
Tara Lin ◽  
Brett Lomenick ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
Preliminary Data ◽  
Lymphoblastic Leukemia ◽  
Parp Cleavage ◽  
Sodium Glutamate ◽  
Alpha Tubulin ◽  
Acetylated Tubulin ◽  
Treat All

Abstract Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. Despite effective chemotherapy, 25 to 30% of children will relapse. In adults, less than 30% of patients with ALL are cured. Therefore, it is critical that we identify novel therapies to treat ALL. We are studying the effects of a small molecule compound known as tubacin (tubulin acetylation inducer) that selectively inhibits histone deacetylase 6 (HDAC6) resulting in increased acetylation of alpha-tubulin by inhibiting one of the two catalytic domains of HDAC6. We found that treatment of both pre-B and T-ALL cell lines with tubacin inhibits growth at very low micromolar concentrations (Jurkat IC50=1μM, Loucy IC50=3μM, REH IC50=2μM, Nalm6 IC50=5μM). We also determined that there is a therapeutic window, since tubacin inhibits the growth of normal bone marrow progenitor cells in methylcellulose colony assays at 20μM and normal human lymphocytes cultured in IL-2 at an IC50 of 16μM. We next tested the effects of tubacin in vivo. SCID mice injected with pre-B ALL Nalm-6 cells were treated with tubacin intraperitoneally at 50 mg/kg/day. Preliminary data using bioluminescence imaging in SCID mouse models showed that tubacin inhibited leukemic progression in vivo. To understand the mechanism of tubacin in ALL cells, we examined both apoptosis and cell cycle regulation by PARP cleavage, activation of caspases, and propidium iodide staining with FACs analysis. Tubacin induced apoptosis of pre-B and T-ALL cells within 12 hours of treatment. There was no effect on cell cycle progression, Retinoblastoma protein phosphorylation, or p21 upregulation, which have been observed with other HDAC inhibitors. Unlike in myeloma cells, tubacin did not increase JunK/SAPK activation or accumulation of acetylated HSP90 in ALL cells. Tubacin treatment resulted in accumulation of acetylated alpha-tubulin after 1 hour and an increase in polyubiquitinated proteins after 7 hours. To address potential mechanisms of tubacin in ALL, we tested whether Na+/K+ ATPase could be contributing to apoptosis. Previous work has shown that treatment with L-glutamate dissociates the Na+/K+ ATPase complex from acetylated tubulin and restores ATPase enzymatic activity. We hypothesized that the accumulation of acetylated tubulin could potentially inhibit the activity of the cytosolic Na/K ATPase pump, which could be reversed by treatment with 1mM sodium glutamate. Preliminary data demonstrate that we can partially rescue the effects of tubacin on PARP cleavage with sodium glutamate. These results suggest that tubacin induces apoptosis through a novel pathway in ALL cells and provide rationale for targeting the aggresome pathway to treat ALL in the future.

scholarly journals Microtubule stabilization specifies initial neuronal polarization

2008 ◽  
Vol 180 (3) ◽  
pp. 619-632 ◽  
Author(s):  
Harald Witte ◽  
Dorothee Neukirchen ◽  
Frank Bradke
Keyword(s):  
Hippocampal Neurons ◽  
Glycogen Synthase ◽  
Initial Step ◽  
Microtubule Dynamics ◽  
Neuronal Polarity ◽  
Low Doses ◽  
Microtubule Stabilization ◽  
Physiological Signal ◽  
Microtubule Stability ◽  
Neuronal Polarization

Axon formation is the initial step in establishing neuronal polarity. We examine here the role of microtubule dynamics in neuronal polarization using hippocampal neurons in culture. We see increased microtubule stability along the shaft in a single neurite before axon formation and in the axon of morphologically polarized cells. Loss of polarity or formation of multiple axons after manipulation of neuronal polarity regulators, synapses of amphids defective (SAD) kinases, and glycogen synthase kinase-3β correlates with characteristic changes in microtubule turnover. Consistently, changing the microtubule dynamics is sufficient to alter neuronal polarization. Application of low doses of the microtubule-destabilizing drug nocodazole selectively reduces the formation of future dendrites. Conversely, low doses of the microtubule-stabilizing drug taxol shift polymerizing microtubules from neurite shafts to process tips and lead to the formation of multiple axons. Finally, local stabilization of microtubules using a photoactivatable analogue of taxol induces axon formation from the activated area. Thus, local microtubule stabilization in one neurite is a physiological signal specifying neuronal polarization.

scholarly journals PKCε Phosphorylates and Mediates the Cell Membrane Localization of RhoA

ISRN Oncology ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-9
Author(s):  
Tizhi Su ◽  
Samuel Straight ◽  
Liwei Bao ◽  
Xiujie Xie ◽  
Caryn L. Lehner ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Cell Membrane ◽  
Cell Invasion ◽  
Time Course ◽  
Time Lapse ◽  
Kinase C ◽  
Phosphorylation Event ◽  
Phosphopeptide Mapping ◽  
First Time ◽  
Initial Peak

Protein kinase Cε (PKCε) signals through RhoA to modulate cell invasion and motility. In this study, the multifaceted interaction between PKCε and RhoA was defined. Phosphopeptide mapping revealed that PKCε phosphorylates RhoA at T127 and S188. Recombinant PKCε bound to recombinant RhoA in the absence of ATP indicating that the association between PKCε and RhoA does not require an active ATP-docked PKCε conformation. Activation of PKCε resulted in a dramatic coordinated translocation of PKCε and RhoA from the cytoplasm to the cell membrane using time-lapse fluorescence microscopy. Stoichiometric FRET analysis revealed that the molecular interaction between PKCε and RhoA is a biphasic event, an initial peak at the cytoplasm and a gradual prolonged increase at the cell membrane for the entire time-course (12.5 minutes). These results suggest that the PKCε-RhoA complex is assembled in the cytoplasm and subsequently recruited to the cell membrane. Kinase inactive (K437R) PKCε is able to recruit RhoA to the cell membrane indicating that the association between PKCε and RhoA is proximal to the active catalytic site and perhaps independent of a PKCε-RhoA phosphorylation event. This work demonstrates, for the first time, that PKCε phosphorylates and modulates the cell membrane translocation of RhoA.

scholarly journals Polarized secretion of tyrosine-sulphated proteins and free tyrosine O-sulphate by filter-grown Madin-Darby canine kidney (MDCK) cells

1991 ◽  
Vol 279 (1) ◽  
pp. 289-295 ◽  
Author(s):  
J R Han ◽  
M C Liu
Keyword(s):  
Time Course ◽  
Mdck Cells ◽  
Time Lapse ◽  
Fresh Medium ◽  
Madin Darby Canine Kidney ◽  
Protein Preparation ◽  
Polarized Secretion ◽  
Darby Canine Kidney ◽  
Canine Kidney

Filter-grown Madin-Darby canine kidney (MDCK) cells labelled for 24 h with [35S]sulphate were found to secrete macromolecules [35S]sulphated on their carbohydrate moieties predominantly into the basolateral medium, whereas the tyrosine-[35S]sulphated proteins synthesized were predominantly secreted into the apical medium. In contrast with the predominant apical secretin of tyrosine-[35S]sulphated proteins, the free tyrosine O-[35S]sulphate (Tyr[35S]) was released mostly into the basolateral medium. A time-lapse study using prelabelled MDCK cells incubated in fresh medium revealed that, during the 48 h time course monitored, the release of tyrosine-[35S]sulphated proteins into the apical medium was faster and quantitatively greater than that into the basolateral medium. During the same time there was a concomitant release, predominantly into the basolateral medium, of the free Tyr[35S] derived from the degradation of tyrosine-[35S]sulphated proteins. An endocytotic degradation experiment was performed to demonstrate the endocytosis of tyrosine-sulphated proteins and their degradation to generate free TyrS. It was found that free Tyr[35S] was generated and released when an apically secreted (or basolaterally secreted) tyrosine-[35S]sulphated protein preparation was added to the apical medium (or the basolateral medium) of unlabelled filter-grown MDCK cells. In both cases, the free Tyr[35S] generated was predominantly released into the basolateral medium similar to the results obtained in the time-lapse study.

Sign in / Sign up

Export Citation Format

Share Document