A new class of fluorescent triazaborolopyridinium compounds was synthesized from hydrazones of 2-hydrazinylpyridine (HPY) and evaluated as potential dyes for live cell imaging applications. The HPY dyes are small, absorption/emission properties are tunable through variation of pyridyl or hydrazone substituents, and offer favorable photophysical characteristics featuring large Stokes shifts and general insensitivity to solvent or pH. The neutral charge, cell membrane permeability and favorable relative influences on the water solubility of HPY conjugates are complementary to existing fluorescent dyes, and offer advantages for the development of receptor-targeted small molecule probes. This potential was assessed through the development of a new class of cysteine-derived HPY-conjugate imaging agents for the kinesin spindle protein (KSP) that is expressed in the cytoplasm during mitosis, and is a promising chemotherapeutic target. Conjugates possessing the neutral HPY or charged AlexaFluor dyes that function as potent, selective allosteric inhibitors of the KSP motor were compared using biochemical and cell-based phenotypic assays, and live-cell imaging. These results demonstrate the effectiveness of the HPY dye moiety as components of probes for an intracellular protein target, and highlight the importance of dye structure in determining the pathway of cell-entry, and the overall performance of small molecule conjugates as imaging agents.

In unicellular fungi, Mob1 facilitates mitotic exit and cytokinesis through its interaction with NDR family kinases. However, its role in regulating cell division in metazoans is less well understood. In this report, we find that Mob1 is required for the recruitment of the chromosomal passenger complex to the spindle midzone during early anaphase.