Structural mechanisms of centromeric nucleation of the kinetochore complex

Descripción del proyecto

La formación del complejo cinetocórico en el centrómero

Cuando una célula se divide en dos, el material genético debe repartirse para que cada célula hija reciba su parte, un proceso fundamental en la división celular, la evolución y el desarrollo de enfermedades. En los organismos eucariotas, como los seres humanos y las levaduras, los cromosomas se unen a los microtúbulos del huso mitótico a través de complejos cinetocóricos, que facilitan la separación del ADN. No se sabe muy bien cómo se forma el complejo cinetocórico interno sobre el ADN centromérico, que es el punto donde los microtúbulos del huso se unen a los cromosomas. En el proyecto CENStruK, que cuenta con el apoyo de las acciones Marie Skłodowska-Curie, se pretende aprovechar el sistema modelo de la levadura con gemación, que posee los cinetocoros más pequeños conocidos, para estudiar los mecanismos de ensamblaje del cinetocoro. La investigación se apoyará en estudios biofísicos y estructurales mediante técnicas avanzadas como la criomicroscopía y la tomografía crioelectrónica.

Objetivo

Ensuring accurate segregation of the genetic material is an essential life process. In eukaryotes, such as humans and yeasts, regions of DNA known as centromeres nucleate the assembly of the megadalton kinetochore complex, which connects to and regulates spindle-microtubules that drive chromosome segregation. While significant progress has been made in the past decade in understanding the structural architecture of the inner kinetochore Ctf19 complex (CCAN in humans), a fundamental gap in knowledge remains concerning how the inner kinetochore is assembled upon native centromeric DNA. This gap is particularly important, as it addresses critical mechanistic steps involved in maintaining genome integrity across cell divisions, evolution, and disease. The budding yeast Saccharomyces cerevisiae is a fundamental model system for studying centromeres and kinetochores, as its kinetochores, the smallest known, represent the organizational module that, through repeated units, form larger kinetochores like those found in humans. In CENStruK, I will combine my background and expertise in yeast genetics and cell biology with the host lab’s expertise in mechanistic biochemistry, its large collection of proteins involved chromosome segregation, and its advanced electron microscopy facilities to fill this gap. CENStruK is divided into two complementary structural approaches. First, by harnessing required protein chaperones, I will reconstitute a key step in yeast’s inner kinetochore assembly that is required for recognition of its native centromere sequence. Second, I will deploy the ‘CentiCEN’ strain, which houses over 100 homogenized centromeres. This strain will be used in parallel for biophysical and structural studies of the endogenous CEN-kinetochore complex using cryo-EM and cryo-ET. My studies will not only shed light on a fundamental unknown in chromosome biology but will also inform future work in diverse eukaryotic species.