Radiating in all directions from the centrosomes at the spindle poles are astral microtubules, which separate the poles and orient the mitotic spindle with respect to other cellular components. Kinetochore microtubules attach to a specialized region within the centromeres of the sister chromatids to assist in maneuvering the chromosomes to the mitotic plate. The polar microtubules overlap in the central region of the spindle and are responsible for pushing the poles (centrosomes) farther apart. On the molecular level, three sets of spindle microtubules can be distinguished. As the spindle grows, the centrosomes begin to translocate to opposite ends of the nucleus, apparently driven by the addition of new tubulin monomers to the existing filament network. The mitotic spindle that begins to form in early prophase is a bipolar structure composed of microtubules and associated proteins. Centromeres are necessary for proper segregation of the sister chromatids during later stages of mitosis. The chromatin in the centromere exhibits a somewhat higher degree of condensation than the rest of the chromosome. Each duplicated chromosome contains two identical sister chromatids joined together along their length, with a constricted region occurring at a specific DNA sequence known as the centromere. The chromatin is stained with a blue fluorescent probe (DAPI), while the microtubule network (mitotic spindle) is stained green (Alexa Fluor 488) and cellular mitochondria are stained with a red dye (MitoTracker Red CMXRos). Presented in the digital fluorescence microscopy image above is a single rat kangaroo ( PtK2) kidney cell in the early stages of prophase. View a second, third, and fourth fluorescence image of prophase. The tubulin monomers are dynamically redirected by the dividing cell to form the main component of the mitotic apparatus, the mitotic spindle, which is bounded by the centrosomes and begins to appear along the periphery of the nuclear membrane. During prophase, major changes also occur in the cytoplasm, including disassembly of the cytoskeleton components based on tubulin (cytoplasmic microtubules). The nucleoli, primarily responsible for the production of ribosomal RNA, begin to disappear as the chromosomes condense. The first stage of mitosis is known as prophase, where the nuclear chromatin starts to become organized and condenses into thick strands that eventually become chromosomes observable in the optical microscope.
Observing Mitosis with Fluorescence Microscopy Prophase Molecular Expressions Cell Biology: Mitosis with Fluorescence Microscopy - Prophase
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