Anaphase
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Anaphase starts when the anaphase promoting complex marks an inhibitory chaperone called securin for destruction by ubiquitylating it. Securin is a protein which inhibits a protease known as separase. The destruction of securin unleashes separase which then breaks down cohesin, a protein responsible for holding sister chromatids together.[2]
Anaphase is characterized by two distinct motions. The first of these, anaphase A, moves chromosomes to either pole of a dividing cell (marked by centrosomes, from which mitotic microtubules are generated and organised). The movement for this is primarily generated by the action of kinetochores, and a subclass of microtubule called kinetochore microtubules.
A combination of different forces have been observed acting on chromatids in anaphase A, but the primary force is exerted centrally. Microtubules attach to the midpoint of chromosomes (the centromere) via protein complexes (kinetochores). The attached microtubules depolymerise and shorten, which together with motor proteins creates movement that pulls chromosomes towards centrosomes located at each pole of the cell.[5]
The second part of anaphase is driven by its own distinct mechanisms. Force is generated by several actions. Interpolar microtubules begin at each centrosome and join at the equator of the dividing cell. They push against one another, causing each centrosome to move further apart. Meanwhile, astral microtubules begin at each centrosome and join with the cell membrane. This allows them to pull each centrosome closer to the cell membrane. Movement created by these microtubules is generated by a combination of microtubule growth or shrinking, and by motor proteins such as dyneins or kinesins.[6]
Anaphase accounts for approximately 1% of the cell cycle's duration.[7] It begins with the regulated triggering of the metaphase-to-anaphase transition. Metaphase ends with the destruction of B cyclin. B cyclin is marked with ubiquitin which flags it for destruction by proteasomes, which is required for the function of metaphase cyclin-dependent kinases (M-Cdks). In essence, Activation of the Anaphase-promoting complex (APC) causes the APC to cleave the M-phase cyclin and the inhibitory protein securin which activates the separase protease to cleave the cohesin subunits holding the chromatids together.
Anaphaseis the fourth phase of mitosis, the process that separates the duplicatedgenetic material carried in the nucleus of a parent cell into two identicaldaughter cells. Before anaphase begins, the replicated chromosomes, calledsister chromatids, are aligned at along the equator of the cell on theequatorial plane. The sister chromatids are pairs of identical copies of DNAjoined at a point called the centromere.
Duringanaphase, each pair of chromosomes is separated into two identical, independentchromosomes. The chromosomes are separated by a structure called the mitoticspindle. The mitotic spindle is made of many long proteins called microtubules,which are attached to a chromosome at one end and to the pole of a cell at theother end. The sister chromatids are separated simultaneously at theircentromeres. The separated chromosomes are then pulled by the spindle toopposite poles of the cell.
The separation of sister chromatids during anaphase is the culmination of mitosis and one of the most strikingly beautiful examples of cellular movement. It consists of two distinct processes: Anaphase A, the movement of chromosomes toward spindle poles via shortening of the connecting fibers, and anaphase B, separation of the two poles from one another via spindle elongation. I focus here on anaphase A chromosome-to-pole movement. The chapter begins by summarizing classical observations of chromosome movements, which support the current understanding of anaphase mechanisms. Live cell fluorescence microscopy studies showed that poleward chromosome movement is associated with disassembly of the kinetochore-attached microtubule fibers that link chromosomes to poles. Microtubule-marking techniques established that kinetochore-fiber disassembly often occurs through loss of tubulin subunits from the kinetochore-attached plus ends. In addition, kinetochore-fiber disassembly in many cells occurs partly through 'flux', where the microtubules flow continuously toward the poles and tubulin subunits are lost from minus ends. Molecular mechanistic models for how load-bearing attachments are maintained to disassembling microtubule ends, and how the forces are generated to drive these disassembly-coupled movements, are discussed.
Lactate is abundant in rapidly dividing cells due to the requirement for elevated glucose catabolism to support proliferation1-6. However, it is not known whether accumulated lactate affects the proliferative state. Here, we deploy a systematic approach to determine lactate-dependent regulation of proteins across the human proteome. From these data, we elucidate a mechanism of cell cycle regulation whereby accumulated lactate remodels the anaphase promoting complex (APC/C). Remodeling of APC/C in this way is caused by direct inhibition of the SUMO protease SENP1 by lactate. We discover that accumulated lactate binds and inhibits SENP1 by forming a complex with zinc in the SENP1 active site. SENP1 inhibition by lactate stabilizes SUMOylation of two residues on APC4, which drives UBE2C binding to APC/C. This direct regulation of APC/C by lactate stimulates timed degradation of cell cycle proteins, and efficient mitotic exit in proliferative human cells. The above mechanism is initiated upon mitotic entry when lactate abundance reaches its apex. In this way, accumulation of lactate communicates the consequences of a nutrient replete growth phase to stimulate timed opening of APC/C, cell division, and proliferation. Conversely, persistent accumulation of lactate drives aberrant APC/C remodeling and can overcome anti-mitotic pharmacology via mitotic slippage. Taken together, we define a biochemical mechanism through which lactate directly regulates protein function to control cell cycle and proliferation.
The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.
The spindle checkpoint prevents chromosome mis-segregation by delaying sister chromatid separation until all chromosomes have achieved bipolar attachment to the mitotic spindle. Its operation is essential for accurate chromosome segregation, whereas its dysregulation can contribute to birth defects and tumorigenesis. The target of the spindle checkpoint is the anaphase-promoting complex (APC), a ubiquitin ligase that promotes sister chromatid separation and progression to anaphase. Using a short hairpin RNA screen targeting components of the ubiquitin-proteasome pathway in human cells, we identified the deubiquitinating enzyme USP44 (ubiquitin-specific protease 44) as a critical regulator of the spindle checkpoint. USP44 is not required for the initial recognition of unattached kinetochores and the subsequent recruitment of checkpoint components. Instead, it prevents the premature activation of the APC by stabilizing the APC-inhibitory Mad2-Cdc20 complex. USP44 deubiquitinates the APC coactivator Cdc20 both in vitro and in vivo, and thereby directly counteracts the APC-driven disassembly of Mad2-Cdc20 complexes (discussed in an accompanying paper). Our findings suggest that a dynamic balance of ubiquitination by the APC and deubiquitination by USP44 contributes to the generation of the switch-like transition controlling anaphase entry, analogous to the way that phosphorylation and dephosphorylation of Cdk1 by Wee1 and Cdc25 controls entry into mitosis.
Analysis of fixed cells using indirect in situ immunofluorescence indicated that only a few clb1Δ clb2-VI cells entered anaphase (Fig. 1B). Instead, spindle disassembly occurred at later time points, indicating that complete CDK inactivation and hence exit from mitosis eventually occur in clb1Δ clb2-VI cells. It is however important to note that exit from mitosis is substantially delayed in clb1Δ clb2-VI cells because they undergo spindle disassembly at least 45 min after wild-type cells (Fig. 1B). In the live-cell analysis, clb1Δ clb2-VI cells behaved slightly differently. After a delay, most cells elongated their spindles and completed anaphase (Fig. 1D; Supplemental Movie 2). The basis for this difference in behavior is at present unknown. It is nevertheless clear that, under both analysis conditions, the onset of anaphase spindle elongation was impaired in clb1Δ clb2-VI cells.
clb1Δ clb2-VI cells are defective in Securin degradation. (A) Wild-type cells carrying Pds1-HA (A1015) and clb1Δ clb2-VI cells carrying Pds1-HA and Scc1-18Myc (A15111) were grown and treated as in Figure 1B. The percentage of cells with metaphase and anaphase spindles (graph) was determined and Pds1-HA protein levels were examined by Western blot analysis. Pgk1 was used as a loading control in Western blots. (B) mad1Δ rad9Δ (A15999), clb1Δ clb2-VI (A15111), and mad1Δ rad9Δ clb1Δ clb2-VI (A16000) cells, each carrying Pds1-HA and Scc1-18Myc, were grown and treated as in Figure 1B. The percentage of cells with metaphase (left graph) and anaphase (right graph) spindles, and the amounts of full-length Scc1-18Myc (Scc1*), the C-terminal cleavage fragment of Scc1-18Myc (cleaved Scc1), and Pds1-HA were examined. Pgk1 was used as a loading control in Western blots. 59ce067264
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In anaphase, the separation of sister chromatids is driven by various cellular components, including microtubules and motor proteins. Similarly, in exterior painting services, skilled professionals utilize a range of tools and techniques to ensure an efficient and effective painting process. This may involve using brushes, rollers, sprayers, and other equipment to apply paint evenly to the exterior surfaces.