Log In Sign up
The largest database of trusted experimental protocols
> Anatomy > Cell Component > Spindle Poles

Spindle Poles

Spindle Poles are specialized structures within eukaryotic cells that play a crucial role in cell division.
They are composed of microtubules and associated proteins, and are responsible for separating chromosomes during mitosis and meiosis.
Spindle Poles ensure accurate chromosomal segregation by forming a bipolar spindle apparatus that attaches to and pulls apart the sister chromatids.
Disruptions in Spindle Pole structure or function can lead to chromosomal instability and potentially contribute to the development of cancer.
Understanding the molecular mechanisms governing Spindle Pole dynamics is an importnat area of cell biology resaerch.

Most cited protocols related to «Spindle Poles»

1
Yeast and E. coli Growth Kinetics
Data for Fig. 4a was gathered using a Tecan Infinity M200 plate reader and a BY4741 strain of Saccharomyces cerevisiae growing in synthetic complete media supplemented with 0.4% glucose and 1% galactose at 30 °C, following an established protocol24 (link). Optical density was measured at an absorbance wavelength of 595 nm every 11.4 min.
Data for Fig. 4b was gathered using a Spectrostar Omega microplate reader and a BW25113 strain of Escherichia coli growing in MM9 (sodium–sodium instead of sodium–potassium) media with 0.1% glucose and 1,106 mOsm sucrose at 37 °C. Optical density was measured at an absorbance wavelength of 600 nm every 7.5 min.
Data for Fig. 5a is from ref. 15 (link).
Data for Fig. 5b was gathered using a custom spinning disk confocal microscope for 20 min in 20 s time steps with 50 ms exposure time per focal plane. Spindle pole bodies were labelled with Spc42-Cerulean. An image stack of 30 z-planes with 300 nm step size was gathered for each time point to allow the position of the spindle poles to be fitted to three-dimensional Gaussian distributions and tracked in time. Imaging, fitting and tracking followed an established protocol16 (link).
Swain P.S., Stevenson K., Leary A., Montano-Gutierrez L.F., Clark I.B., Vogel J, & Pilizota T. (2016). Inferring time derivatives including cell growth rates using Gaussian processes. Nature Communications, 7, 13766.
Full text: Click here
Publication 2016
Escherichia coli Galactose Glucose M-200 Microscopy, Confocal Potassium Saccharomyces cerevisiae Sodium Spindle Pole Body Spindle Poles Strains Sucrose Vision
2
Quantitative Spindle Angle Analysis
To measure spindle angles (see Fig. 4 A for schematic), confocal images of metaphase cells in the middle region of the cysts were collected, and the centroid of the cyst (Fig. 4 A, dark blue circle) and center of the spindle axis (Fig. 4 A, pink circles) were drawn using ImageJ (National Institutes of Health). The angle (Fig. 4 A, red) between the spindle axis (Fig. 4 A, black lines) and the line connecting the centroid of the cyst and the center of the spindle (Fig. 4 A, dashed lines) was analyzed. When both spindle poles were not in one z section, three z sections including each spindle pole were taken and merged to draw a line of spindle axis.
Jaffe A.B., Kaji N., Durgan J, & Hall A. (2008). Cdc42 controls spindle orientation to position the apical surface during epithelial morphogenesis. The Journal of Cell Biology, 183(4), 625-633.
Publication 2008
Cells Cyst Epistropheus Metaphase Spindle Poles
3
Quantitative Analysis of Cdc27 Spindle Localization
HeLa cells were transfected with control or indicated siRNA (ON-TARGETplus SMARTpool siRNA, Dharmacon) for 48 h, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100/PBS, and stained with 0.5 μg/ml Hoechst 33342, rat anti-α-tubulin (Serotec, Oxford, United Kingdom) and either rabbit anti-Cdc27, Eg5, or cyclin B or mouse anti-centrin. For acute OA treatment, cells were treated with 175 nM OA for 13 min before fixation and staining. Slides were mounted with ProLong Gold anti-fade reagent (Invitrogen), and projection images (10-μm stacks captured every 0.5 μm) were captured with a Zeiss Axio Imager.Z1 microscope (Thornwood, NY) equipped with a CoolSNAP HQ camera (Photometrics, Tucson, AZ) and operated with SlideBook 4.2 (Intelligent Imaging, Denver, CO) at 63× (NA 1.4) at room temperature. One hundred cells were analyzed to determine the percentage of cells with Cdc27 spindle pole localization in control, indicated siRNA, and acute OA-treated cells. A 2 × 2-μm square was drawn around each of 20 spindle poles from control, indicated siRNA, or acute OA-treated cells, and the mean fluorescence intensity of Cdc27 or cyclin B spindle pole staining was plotted as arbitrary units (AU). Additionally, intensity measurements were taken along an axis intersecting the two spindle poles and the fluorescence intensity was graphed as arbitrary units (AU). Image processing was performed using Adobe Photoshop CS2 (version 9.0.2; San Jose, CA).
Torres J.Z., Ban K.H, & Jackson P.K. (2010). A Specific Form of Phospho Protein Phosphatase 2 Regulates Anaphase-promoting Complex/Cyclosome Association with Spindle Poles. Molecular Biology of the Cell, 21(6), 897-904.
Publication 2010
alpha-Tubulin Cells Cyclin B Epistropheus Fluorescence Gold HeLa Cells HOE 33342 Microscopy Mus paraform Rabbits RNA, Small Interfering Spindle Poles Trimethoprim-Sulfamethoxazole Combination Triton X-100
4
Immunofluorescence Labeling of Cellular Cytoskeleton
After the above fixation methods, cells were incubated in a primary antibody solution prepared in 1% BSA and Banker’s PBS-Tx (different concentrations of Triton X-100 were used depending on the experiment) overnight at 4 °C. Primary antibodies used, included rabbit anti-β-tubulin (1:1000, Abcam, ab6046), mouse anti-acetylated α-tubulin (1:50,000, Sigma, clone 6-11B-1), rabbit anti-detyrosinated tubulin (1:500, Millipore, AB3201), mouse anti-polyglutamylated tubulin (1:500, Sigma, T9822), rabbit anti-adenylyl cyclase 3 (AC3, 1:200, Santa Cruz Biotechnology, sc-588), rabbit anti-ADP-ribosylation factor-like protein 13b (Arl13b, 1:200, UC Davis/NIH NeuroMab Facility clone N295B/66), rabbit anti-centrosome and spindle pole associated protein 1 (CSPP1, 1:200, Proteintech, 11931-1-AP), rabbit anti-intraflagellar transport protein 20 (IFT20, 1:200, Sigma, HPA021376), rabbit anti-intraflagellar transport protein 88 (IFT88, 1:200, Proteintech, 13967-1-AP), and mouse anti-Golgi matrix protein 130 (GM130, 1:1,000, BD Bioscience, 610822). After extensive washing, cells were then incubated in either mouse or rabbit Alexafluor 488 and Alexafluor 546 secondary antibodies (1:500, Life Technologies) for 1 h at room temperature. Last, nuclei were labeled with Hoechst 33258 (1 µg/ml), and actin was labeled with phalloidin-546 (1:200, Sigma, A22283) when appropriate. All coverslips were mounted with Fluoromount G (Southern Biotech, 0100-01). Images were obtained with a Zeiss AxioImager.Z1 upright microscope equipped with an AxioCam MRm camera, using a 63× plan-apochromat (1.4 NA) oil objective (Zeiss) and fluorescent filter sets 20, 34, 38HE, and 50 (Zeiss). Images were processed with AxioVision Rel. 4.5 software, and imported into ImageJ [36 (link)] and/or Adobe Photoshop CS6 version 13.0 × 64 to assemble montages.
Hua K, & Ferland R.J. (2017). Fixation methods can differentially affect ciliary protein immunolabeling. Cilia, 6, 5.
Full text: Click here
Publication 2017
Actins Adenylate Cyclase ADP-Ribosylation Factors alpha-Tubulin Antibodies Carrier Proteins Cell Nucleus Cells Centrosome Clone Cells Golgi Matrix Proteins Hoechst 33258 Immunoglobulins Microscopy Mus Phalloidine Proteins Rabbits Spindle Poles Synapsin I Triton X-100 Tubulin
5
Microscopic Imaging of Cellular Structures
For analysis of fixed samples mounted in PPDM (90% glycerol, 0.5% p-phenylenediamine, 20 mM Tris-HCl pH 8.8), images were acquired on a DeltaVision deconvolution Olympus IX70 microscope (Applied Precision) equipped with a CoolSnap CCD camera (Roper Scientific) at 20°C using a 100×, 1.35 NA Olympus U-Planapo oil objective lens. Immunofluorescence of fixed embryos was performed as described (Desai et al., 2003 (link)), using the following rabbit antibodies at a concentration of 1 μg/ml: α–CAR-1 (Cy3-labeled; described above); α–AIR-2 (Cy-5 labeled; generated against a GST fusion to the full-length protein); α–ZEN-4 (Cy-5 labeled; generated against a GST fusion to the COOH-terminal 108 aa); the mouse monoclonal antibody DM1α (Oregon green 488–labeled; Sigma-Aldrich); the goat polyclonal GFP antibody (Oregon green 488–labeled; generated against a 6x-histidine fusion to the full- length protein); and the unlabeled rat CGH-1 antibody (JDCR5; a gift of K. Blackwell, Joslin Diabetes Center, Boston, MA). For analysis of gonads, the tails of adult hermaphrodites were amputated in 5% sucrose and 100 mM NaCl to extrude the gonads. Fixation and immunofluorescence on gonads was performed as described for embryos. For live analysis, embryos were mounted on agarose pads as described previously (Oegema et al., 2001 (link)), and imaged on a spinning disc confocal microscope (Nikon Eclipse TE2000-E) equipped with a Hamamatsu Orca-ER CCD camera at 20°C using a Nikon 60×, 1.4 NA Planapo oil objective lens. For osmotically sensitive embryos and embryos imaged in the absence of compression, filming was performed in a depression slide containing meiosis media (25 mM Hepes at pH 7.4, 60% Leibowitz L-15 Media, 20% FBS, 500 μg/ml inulin) and sealed with petroleum jelly. Analysis of spindle pole separation, spindle microtubule density, and furrow movement was performed using Metamorph software.
Kymographs were constructed by compressing the image of the furrow region from each time point (same region as in Fig. 5 B) to a single vertical line, in which the maximum intensity along the x-axis of each original image is displayed for each point along the y-axis. The vertical strips for sequential time points are laid adjacent to each other so that time increases from left to right along the x-axis.
Audhya A., Hyndman F., McLeod I.X., Maddox A.S., Yates JR I.I.I., Desai A, & Oegema K. (2005). A complex containing the Sm protein CAR-1 and the RNA helicase CGH-1 is required for embryonic cytokinesis in Caenorhabditis elegans. The Journal of Cell Biology, 171(2), 267-279.
Publication 2005
Adult Antibodies Diabetes Mellitus Embryo Epistropheus Extrude Fluorescent Antibody Technique Glycerin Goat Gonads HEPES Hermaphroditism Histidine Immunoglobulins Inulin Kymography Lens, Crystalline Meiosis Mice, House Microscopy Microscopy, Confocal Microtubules Monoclonal Antibodies Movement Orcinus orca Oregon Green 488 carboxylic acid Petrolatum Phenylenediamines Proteins Rabbits Sepharose Sodium Chloride Spindle Poles Sucrose Tail Tromethamine

Most recents protocols related to «Spindle Poles»

1
SpinX: Automated Spindle Tracking and Analysis
Fig. S1 describes data composition, Fig. S2 details SpinX pipelines for automated label generation, Fig. S3 evaluates automated label generation using SpinX, Fig. S4 explains data augmentation techniques, Fig. S5 evaluates two SpinX models using IoU metric, Fig. S6 evaluates two SpinX models using Loss of function metric, Fig. S7 are example segmentations from different architectures, Fig. S8 evaluates SpinX Stage 3 for accuracy manually, Fig. S9 on cell cortex eccentricity, Fig. S10 presents spindle length and width measured through SpinX, Fig. S11 outlines analytical solution for 3D Ray-tracing, Fig. S12 compares Refined and Old SpinX algorithms for recording spindle pole positions, Fig. S13 shows increased spindle rotation following CENP-E inhibition, Fig. S14 shows no increase in spindle rotation following MARK2 inhibition, and Fig. S15 presents change in MARK2-YFP localization following its inhibition. Video 1 summarizes SpinX spindle and cortex tracking features. Table S1 shows comparison of SpinX with previous software for spindle and cell cortex detection and tracking. Table S2 shows differences between SpinX-base and SpinX-optimized. Table S3 shows evaluation of SpinX-base and SpinX-optimized models. Table S4 shows evaluation of annotation. Table S5 shows spindle tracking evaluation. Table S6 shows parameters used for PSF simulation.
Dang D., Efstathiou C., Sun D., Yue H., Sastry N.R, & Draviam V.M. (2023). Deep learning techniques and mathematical modeling allow 3D analysis of mitotic spindle dynamics. The Journal of Cell Biology, 222(5), e202111094.
Full text: Click here
Publication 2023
Cells Cortex, Cerebral MARK2 protein, human Psychological Inhibition Spindle Poles
2
Spindle Pole Position Refinement
The spindle pole refinement algorithm takes initial (x,y,z) spindle pole predictions as an input. The spindle boundary coordinates are obtained by taking the maximum projection of the spindle mask. Then, 3D coordinates are extracted along the spindle length axis (pole-to-pole axis) to obtain the corresponding pixel values. The true spindle pole is defined as the first and last occurrence of positive values in the resulting 1D array. Finally, the new position of spindle poles was updated across all data frames for further calculations (Table 1). Manual analysis used for evaluating the performance of SpinX’s pole position recording was performed on Fiji (ImageJ) software (Schindelin et al., 2012 (link)).
Dang D., Efstathiou C., Sun D., Yue H., Sastry N.R, & Draviam V.M. (2023). Deep learning techniques and mathematical modeling allow 3D analysis of mitotic spindle dynamics. The Journal of Cell Biology, 222(5), e202111094.
Full text: Click here
Publication 2023
Epistropheus Reading Frames Spindle Poles
3
Quantifying Cortical Mud Protein Distribution
We used FIJI to perform our quantifications. Starting at the center of the basolateral region (which we defined as 0), a freehand line was drawn around the perimeter of the cell and pixel intensity determined at each point along the line. Background signal, measured in the cytoplasm, was subtracted from these points. Intensities were normalized by dividing the median intensity and positions were normalized to a line with a length of 360. The graphical representations show data points from multiple cells (as indicated in the legend) using a floating 10‐point average. Measurement of Mud intensity was sometimes complicated by the proximity between spindle poles, which are highly Mud‐positive, and the cortex, which is less so. In these instances, the overlapping region was excluded from the measurement. These exclusions help to explain why Mud intensity profiles are not as smooth as those measured for other proteins.
Neville K.E., Finegan T.M., Lowe N., Bellomio P.M., Na D, & Bergstralh D.T. (2023). The Drosophila mitotic spindle orientation machinery requires activation, not just localization. EMBO Reports, 24(3), e56074.
Publication 2023
Cells Cortex, Cerebral Cytoplasm Perimetry Proteins Spindle Poles
4
Mitotic Spindle Angle Measurement
Spindle angles were determined using FIJI by finding the distance between the mitotic spindle poles: (i) in the xy axis of the flattened ventrolateral ectoderm in a sum projection of the confocal z stack; (ii) in z axis by determining the z planes that the 2 poles appear. The mitotic spindle orientation angle, φ, was calculated by taking the inverse tan of the difference in z depth divided by the distance in xy between the spindle poles (Fig EV3C and D).
Neville K.E., Finegan T.M., Lowe N., Bellomio P.M., Na D, & Bergstralh D.T. (2023). The Drosophila mitotic spindle orientation machinery requires activation, not just localization. EMBO Reports, 24(3), e56074.
Publication 2023
Ectoderm Epistropheus Mitotic Spindle Apparatus Spindle Poles
5
Spindle Angle Determination Protocol
Spindle angle determination was performed as previously described (Finegan et al, 2018 (link)). Angles were determined by drawing a first line connecting either the two spindle poles (if applicable) or along the spindle and a second line along the apical surface of the tissue, then measuring the angle between them. Spindle angle values for control w1118 are reused between Figs 1D, 3F, 4C, 5C and 6F, and for pinsp62/pinsp62 between Figs 3F, 4C and 5C.
Neville K.E., Finegan T.M., Lowe N., Bellomio P.M., Na D, & Bergstralh D.T. (2023). The Drosophila mitotic spindle orientation machinery requires activation, not just localization. EMBO Reports, 24(3), e56074.
Publication 2023
Figs Spindle Poles Tissues

Top products related to «Spindle Poles»

1
Tcs sp5 confocal microscope by Zeiss
Sourced in Germany
The TCS SP5 is a high-performance confocal microscope designed for advanced imaging applications. It features a modular architecture that allows for customization and integration of various detection systems. The TCS SP5 provides users with a versatile platform for performing detailed analysis of samples at the cellular and sub-cellular level.
2
Prolong gold antifade reagent by Thermo Fisher Scientific
Sourced in United States, Germany, Japan, United Kingdom, Italy, Australia, Canada, France, Spain, China
ProLong Gold antifade reagent is a water-based mounting medium designed to retard the photobleaching of fluorophores in fluorescence microscopy applications. It is formulated to provide long-term preservation of fluorescent signals in fixed and immunolabeled samples.
3
Apotome 2 by Zeiss
Sourced in Germany, United States, United Kingdom
The ApoTome.2 is a structured illumination microscopy system developed by Zeiss. It is designed to improve image quality by reducing out-of-focus light, thereby enhancing the contrast and resolution of images captured with a widefield fluorescence microscope.
4
Eclipse te2000 e microscope by Nikon
Sourced in United States, Japan, Italy
The Eclipse TE2000-E is a research-grade inverted microscope designed for a wide range of applications. It features a modular design, allowing for customization with various accessories and options. The core function of the Eclipse TE2000-E is to provide high-quality imaging and observation capabilities for users in laboratory and research settings.
5
Hoechst 33342 by Merck Group
Sourced in United States, Germany, Italy, United Kingdom, Japan, China, France, Sao Tome and Principe, Canada, Macao, Belgium, Israel, Spain, Australia, Poland, Switzerland, India, Denmark, Austria, Netherlands
Hoechst 33342 is a fluorescent dye that binds to DNA. It can be used to visualize and quantify nucleic acids in various applications such as flow cytometry, fluorescence microscopy, and nucleic acid staining.
6
Matlab by MathWorks
Sourced in United States, United Kingdom, Germany, Canada, Japan, Sweden, Austria, Morocco, Switzerland, Australia, Belgium, Italy, Netherlands, China, France, Denmark, Norway, Hungary, Malaysia, Israel, Finland, Spain
MATLAB is a high-performance programming language and numerical computing environment used for scientific and engineering calculations, data analysis, and visualization. It provides a comprehensive set of tools for solving complex mathematical and computational problems.
7
Multiphysics 4 by Comsol
Sourced in United States, Sweden
COMSOL Multiphysics 4.3 is a software platform for modeling and simulating physics-based problems. It provides a comprehensive environment for solving complex problems involving multiple physical phenomena, including structural mechanics, fluid dynamics, heat transfer, and electromagnetics.
8
Uplanapo objective by Olympus
Sourced in Japan, Germany
The UPlanApo objective is a high-performance optical lens designed for microscopy applications. It is engineered to provide high-resolution, aberration-corrected imaging across a wide field of view. The objective's core function is to effectively gather and focus light to create a clear, detailed image of the specimen under observation.
9
86000 sedat quadruple filter set by Chroma Technology
Sourced in United States
The 86000 Sedat Quadruple Filter Set is a lab equipment product designed for researchers and scientists. It consists of four individual filters that can be used for various applications in the laboratory. The core function of this product is to provide controlled light filtration and isolation for experimental purposes. No further details or interpretations about the intended use of this product can be provided in an unbiased and factual manner.
10
Ix70 inverted microscope by Olympus
Sourced in Japan, United States, Germany, United Kingdom
The Olympus IX70 is an inverted microscope designed for biological and life science research applications. It features a stable and ergonomic design, supporting a range of imaging techniques and accessories to facilitate various microscopy workflows.

More about "Spindle Poles"

Spindle poles, also known as centrosomes or microtubule organizing centers (MTOCs), are essential structures within eukaryotic cells that play a crucial role in cell division.
These specialized organelles are composed of microtubules and associated proteins, responsible for separating chromosomes during mitosis and meiosis.
The spindle pole apparatus forms a bipolar spindle that attaches to and pulls apart the sister chromatids, ensuring accurate chromosomal segregation.
Disruptions in spindle pole structure or function can lead to chromosomal instability, potentially contributing to the development of cancer.
Understanding the molecular mechanisms governing spindle pole dynamics is a critical area of cell biology research.
Techniques such as confocal microscopy, using instruments like the TCS SP5 or Eclipse TE2000-E, can provide insights into the spatial and temporal dynamics of spindle poles during cell division.
Fluorescent dyes like Hoechst 33342 can be used to visualize chromosomes, while software like MATLAB and COMSOL Multiphysics 4.3 can be employed for computational modeling and analysis.
Spindle pole research also involves the use of specialized reagents and tools, such as the ProLong Gold antifade reagent and the ApoTome.2 system, which enhance the quality and resolution of fluorescence microscopy images.
Additionally, high-quality optics, such as the UPlanApo objective, and filter sets like the 86000 Sedat Quadruple Filter Set, can improve the detection and analysis of spindle pole dynamics.
By leveraging these advanced techniques and tools, researchers can gain a deeper understanding of the intricate mechanisms underlying spindle pole function and its role in ensuring the fidelity of cell division.
This knowledge can ultimately lead to the development of novel therapeutic strategies for addressing chromosomal instability and related diseases, including cancer.