Sometimes in the life sciences, it’s really tempting to look at a process like mitosis and think, “Wow, we really understand that,” and stop asking questions. In other cases, significant barriers like reliable expression of a non-endogenous reporter in an organ such as the brain is a challenge that seems impossible to overcome. However, those are precisely the moments when science and scientists need to push the boundaries of what we can see. In this post, I discuss two studies where researchers are literally seeing what could not be seen before by developing new methods for looking at their model systems.
In both papers discussed here, the HaloTag® protein and ligands were key to developing new methods allowing scientists to visualize biological phenomena that had been previously inaccessible. HaloTag® protein allows different functionalities to be linked onto a single genetic construct, rapidly and covalently under physiological conditions for experiments ranging from live-cell imaging to capture of protein interaction complexes (1). In the first study, HaloTag-CENP-A + JF635 ligand was the specific tool that let them directly see, in real time, that chromosome-attached microtubules pivot as the spindle elongates. In the second study, HaloTag was used to create a protocol for PET imaging of reporter gene systems in the brain.
Study 1: Reimagining the Way Polar Chromosomes Migrate During Mitosis
The process of mitosis is taught in the earliest biology classes: from the breakdown of the nuclear envelope to the formation of the mitotic spindle and the alignment of the chromosome pairs at the metaphase plate, to the separation of the chromosomes and formation of the two new cell nuclei. Because it has been so well described, it’s easy to think that we know all there is to know about this process and that there are no more questions to ask.
Just last year I reviewed a paper that established a critical role for RNA methylation in spindle assembly. Now, a paper published in 2026 (2) looks at the physical migration of chromosomes during mitosis, focusing on the problem of how cells get “polar” chromosomes (captured outside or “behind” the poles of the spindle) to the metaphase plate.
Researchers have observed in numerous studies that unaligned (behind the spindle pole) and misaligned (near the pole) chromosomes are a source of segregation errors, often ending up micronuclei, correlating with chromosomal instability and cancer-causing potential. Mis- and unaligned chromosomes have been described in several cancer cell lines including ovarian, bone, colorectal and breast cancers and in patient samples of primary metastatic breast cancer. The 1q arm of Chromosome 1 is often associated with cancer development and transformation, and it is suggested that this chromosome is often located behind the pole and at risk of unalignment.
To understand how polar chromosomes segregate, Koprivec and colleagues (1) used super resolution microscopy in retinal pigment epithelial cells with labeled centromeres and either labeled tubulin or centrosomes. Specifically, they used HaloTag® technology as a fluorescent labeling system to visualize kinetochores (HaloTag fused to CENP-A, a centromere protein) in live-cell imaging experiments. RPE1 cells were labeled with HaloTag-CENP-A, and incubated cells for 30 minutes with Janelia Fluor® 635 HaloTag® ligand. Their studies supported a model in which spindle elongation drives repositioning of the polar chromosomes. They tracked the polar kinetochores and their microtubules, as well as the centrosomes and chromosome arms through a confocal microscopy protocol with adjusted pixel size to image RPE1 cells expressing EYFP-α-tubulin with added HaloTag-CENP-A. The kinetochores of a polar chromosome were adjacent to a single astral microtubule (or a small bundle), which pivoted around the centrosome toward the spindle surface while maintaining its connection to the chromosome, as the centrosome moved outward.
Study 2: Quantitative Imaging of Gene Expression in the Brain via PET
The blood-brain barrier is formidable, so getting therapeutics into the central nervous system is difficult at best. Getting an exogenous probe to measure reporter gene expression in the central nervous system is no less difficult. Add to that, the challenge of how pathologies may be affecting gene expression, and you have a truly difficult problem to solve for drug discovery and even basic research aimed at understanding the best ways to target neuropathologies.
In the second study that I discuss here, the researchers developed a HaloTag ligand to help reveal biology in ways previously unavailable. Stolz and colleagues (3) engineered a small-molecule HaloTag® ligand labeled with fluorine-18, designed with a small size and moderate lipophilicity to cross the blood-brain barrier, solving one of the problems for measuring reporter gene expression (RGS) in the brain. They validated the new tracer, called [18F]FB-HTL, first in a HaloTag®-expressing human cell line, next in mice using a viral vector, and finally again in mice, showing that this RGS could detect physiological protein expression in transgenic mice.
The authors conclude that this reporter system offers a versatile, non-invasive tool for longitudinal monitoring of gene expression in the brain, with potential applications in gene therapy validation, neuronal tracing, and models of neurological disease.
Summary
Both of these papers ask questions that couldn’t be answered until scientists developed new ways of seeing biological phenomena. Whether the question was how chromosomes segregate or how to measure gene expression in the brain, HaloTag gave researchers a way to see what they couldn’t see before. In one study, new understanding of a well-studied process was achieved; in the second, a tool now exists to fuel questions and answers around neurological disease research.
Products Used in the Studies
HaloTag® Alexa Fluor 488 Ligand
HaloTag® Janelia Fluor 635 Ligand
pHTN HaloTag CMV-neo Vector (from reference 78 in the study reviewed: Evidence for a HURP/EB free mixed-nucleotide zone in kinetochore-microtubules | Nature Communications )
Literature Cited
Los, G.V. et al. (2008) HaloTag: A novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3(6):373–82. https://doi.org/10.1021/cb800025k
Koprivec, I. et al. (2026) Polar chromosomes are rescued from missegregation by spindle elongation-driven microtubule pivoting https://doi.org/10.1038/s41467-026-69830-1
Stotz S. et al. (2026) A PET reporter ligand for quantitative imaging of gene expression in the brain. Nature Biomedical Engineering. https://doi.org/10.1038/s41551-026-01748-x
This article was conceived and written by a human, reviewed using AI assistance, and edited by a human.
Michele Arduengo, PhD
