Phosphor Imaging with the Azure Sapphire in an Investigation of Chromosome Segregation

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New insight into microtubule-kinetochore interactions

Work recently published in the Journal of Cell Biology1 provides new insight into a fundamental processes of cell biology, chromosome segregation. Flores et al investigated the importance of three specific points of interaction between Dam1 and Ndc80 to the strength and persistence of attachment between the kinetochore and microtubule tips.

This research group previously identified the three regions of interaction. Most recently, they mutated or phosphorylated amino acids in each of the three regions and used laser trapping to assess the effect of the mutations on the persistence of the interaction under constant force and on the rupture strength of the interaction when the applied force was rapidly increased.

Autoradiograph of an SDS-PAGE gel using phosphorimaging from Azure Sapphire
S3. Analysis of phosphorylation levels on wild-type and mutant Dam1 complexes. (A) Autoradiograph of an SDS-PAGE gel (8–14%) showing incorporation of [γ-32P]ATP into different interaction regions of the Dam1 complex. (B) Coomassie blue–stained SDS-PAGE gel (8–14%) of wild-type Dam1 complex with GST-Sli15554–698 and GST-Ipl1p (wild-type), GST-Sli15554–698 alone, GST-Ipl1p alone, and GST-Sli15554–698 together with GST-Ipl1p. Licensed under CC BY 4.0.

Phosphorylation using radioactive 32P-ATP

To measure the extent of phosphorylation of Dam1 proteins, the authors carried out a phosphorylation assay using radioactive 32P-ATP. Radio-labeled products were detected on dried SDS-PAGE gels by phosphor imaging on an Azure Sapphire Biomolecular Imager (Figure S3-A.)

Since the release of this publication, the Azure Sapphire has been succeeded by the new Azure Sapphire FL, which was designed to be the flexible choice in bringing precise quantitation of nucleic acids and proteins. Learn more.

Sapphire FL biomolecular imager
The new Sapphire FL images phosphor screens with high sensitivity. With customizable and user-changeable laser and filter modules, it easily adapts to a lab’s changing needs and advancing research.

The results of the experiments showed that all three regions contribute to the strength and persistence of attachments between the microtubule tips and kinetochores. Two of the regions also contributed to the rupture strength of the interaction. However, phosphorylation of one site had no effect on rupture strength but decreased attachment lifetime. The authors hypothesize these differential effects on long-term stability vs short-term strength could play a role in error correction in vivo.

What happens when a cell divides

When a cell divides, it is essential that each daughter cell receives a complete set of chromosomes. During mitosis, chromosomes condense and become attached to the ends of microtubules that make up the mitotic spindle. As the cell prepares to divide, the spindle microtubules shorten and drag the attached chromosomes to opposite ends of the dividing cell.

Phases of Mitosis
Phases of Mitosis

Incorrect chromosome segregation is usually lethal since the daughter cells will not receive a complete set of genomic information. In multicellular organisms, chromosome mis-segregation is associated with cancer.

How exactly the chromosomes attach to the spindle microtubules is unknown. The tips of the microtubules attach to a large multi-protein structure attached to each chromosome called the kinetochore. However, the microtubules change length by adding or removing tubulin subunits to the tips so important questions remain including how a chromosome attaches to a dynamic structure, as well as how a chromosome remains attached to a microtubule under force while being pulled towards the spindle pole.

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How do kinetochores attach to the microtubule tips?

A protein complex called Ndc80 is required to keep chromosomes attached to the spindle. Experiments in yeast have demonstrated that all four subunits of Ndc80 are essential. In yeast, Ndc80 interacts with another multiprotein complex, Dam1. Dam1 appears to form rings around microtubules and to help the kinetochore-spindle attachment bear load and remain attached while the microtubule changes length.

How do kinetochores attach to the microtubule tips?

The Aurora B kinase is required for accurate chromosome segregation. Phosphorylation of targets by this kinase causes kinetochore-microtubule attachments to release. It is hypothesized that incorrect attachments are not under tension and that the kinase recognizes these non-functional attachments and phosphorylates target sites to break the attachment.

In addition to phosphor imaging, the Sapphire Biomolecular Imager provides densitometry, multichannel fluorescence, near-infrared, chemiluminescence, and white light imaging of blots, gels, tissues, and more. Learn more about the Sapphire Imager and how Azure can support your research by clicking here.
  1. Flores RL, Peterson ZE, Zelter A, et al. Three interacting regions of the Ndc80 and Dam1 complexes support microtubule tip-coupling under load. J Cell Biol. 2022;221(5):e202107016

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