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Two SPB components (Spc42-mTurquoise2 and Cnm67-YFP) previously shown to exhibit high FRET gave 11.0 ± 1.0% (n = 159) FRET efficiency in our system (Muller et al. 2005).
Examination of a GFP-tagged SPB component, Sid4 [ 43], clearly revealed two SPB signals in cells containing monopolar spindles, indicating that the SPBs had duplicated.
Recent studies have identified two SPB components, the pericentrin-like Pcp1 and Csi1, as recruiters of the Alp7-Alp14 complex to the SPB [ 63, 64].
In early S-phase, the SPB are duplicated and during metaphase the two SPB send out microtubules which attach to the chromosomes in a stochastic manner [5], [6].
The total percentage of cells with two SPB foci that contained two GFP foci was calculated for all frames for each strain.
At the metaphase-anaphase transition, the spindle begins to elongate and the two SPB dots move quickly away from each other.
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At t = 50 s, the elastic constant of the spring connecting the two SPBs is then set to zero and its resting length reduced to Δ = 1 μm less than the SPB's resting separation.
At 45 min or 75 min after release from the G1 block most tub4-S74E tub4-S360D360D cellshoweded two SPBs separated by 1 2 µm (Figure 5C, E and Figure S6C, E).
Very rarely centromeres stayed between two SPBs and distant from them.
The two SPBs are probably equally functional in mps1-as1 cells, at least under these experimental conditions.
One explanation is that one of two SPBs is defective in organizing microtubules that extend to capture kinetochores.
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