ABSTRACT
It is known that microtubule-binding proteins including the Ska1 complex and the DNA replication licensing factor, Cdt1, enable the kinetochore-localized Ndc80 complex to form robust kinetochore-microtubule attachments. However, it is not clear how the Ndc80 complex is stably coupled to dynamic spindle microtubule plus-ends. Here, we have developed a conditional auxin-inducible degron approach to reveal a function for Cdt1 in chromosome segregation and kinetochore-microtubule interactions that is separable from its role in DNA replication licensing. Further, we demonstrate that a direct interaction between Cdt1 and Ska1 is required for recruiting Cdt1 to kinetochores and spindle microtubules. Cdt1 phosphorylation by Cdk1 kinase is critical for Ska1 binding, kinetochore-microtubule attachments, and mitotic progression. Furthermore, we show that Cdt1 synergizes with Ndc80 and Ska1 for microtubule binding, including forming a diffusive, tripartite Ndc80-Cdt1-Ska1 complex that can processively track dynamic microtubule plus-ends in vitro. Taken together, our data identify the Ndc80-Cdt1-Ska1 complex as a central molecular unit that can promote processive bidirectional tip-tracking of microtubules by kinetochores.
Subject(s)
Cell Cycle Proteins , Chromosomal Proteins, Non-Histone , Kinetochores , Microtubule-Associated Proteins , Nuclear Proteins , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromosome Segregation , Kinetochores/metabolism , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Mitosis , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolismABSTRACT
Purpose: To evaluate the efficacy of slit lamp breath shields to prevent droplet spray from a simulated sneeze. Design: Experimental study to test the effectiveness of personal protective equipment. Methods: The nozzle of a spray gun was adjusted to angularly disperse a mist of colored dye that approximated a patient sneezing on a dimensionally accurate cardboard slit lamp model. The designs of 6 commercially available breath shields and 1 breath shield repurposed from a plastic container lid were tested. Each breath shield was sprayed in a standardized fashion 3 times, and the amount of overspray was compared to spray with no shield and quantified. The surface area that was sprayed was calculated using a commercially available software with color range function. The average percentage of overspray of each breath shield was computed in comparison to the control. Results: The breath shields ranged in surface area from 116 to 924 cm2, and the amount of overspray varied from 54% to virtually none. Larger breath shields offered better protection than smaller ones. Breath shields attached to the objective lens arm were better barriers than those of comparable size hung by the oculars. A repurposed plastic lid breath shield, 513 cm2, was slightly curved toward the examiner's face and allowed only 2% overspray. The largest breath shield (924 cm2) hung near the oculars and prevented essentially all overspray. Conclusions: The performance of different designs of breath shields was variable. Even high-functioning shields should be used in conjunction with personal protective equipment including masks, goggles, and gloves and handwashing. Ideally patients should also wear a face mask during all slit lamp examinations.
