CenSpark650 Probe for Live Imaging of Centrioles and Cili

CenSpark650, a dual-ligand fluorescent probe for centrioles and cilia selective imaging.

Visualizing centrioles and cilia in live cells was only available through complex genetic engineering. To overcome this limitation, we are proud to announce the upcoming release of CenSpark650, a transformative small-molecule probe designed for the selective visualization of centriolar and axonemal microtubules.

CenSpark650 exploits a unique structural feature: the juxtaposition of inner and outer microtubule-binding sites found exclusively in the microtubule triplets and doublets of centrioles and cilia.  CenSpark650 achieves unprecedented selectivity in both live and fixed specimens across diverse eukaryotic systems.

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Centrosomes are non-membrane-bound organelles that serve as the primary microtubule-organizing centers (MTOCs) in cells. At the core of each centrosome lies a pair of barrel-shaped structures called centrioles, which are positioned perpendicular to one another. Each centriole is meticulously constructed from nine triplets of microtubules arranged in a distinct symmetrical ring. Surrounding this pair is the pericentriolar material (PCM), a dense protein matrix responsible for nucleating and anchoring microtubule networks. During cell division, the centrosome duplicates precisely once to form the two poles of the mitotic spindle, ensuring chromosomes separate equally. Beyond division, the older "mother" centriole can migrate to the cell surface and transform into a basal body, acting as the structural template necessary to grow cilia and flagella. Visualizing centrosomes, centrioles and cilia typically requires fixation and immunostaining while live cell imaging heavily relies on complex genetic tagging of fluorescent proteins or self labelling tags. This is not always compatible with models where genetic engineering is difficult or impossible, thereby making live cell centriole visualization a considerable time and cost intensive task.

CenSpark650 is a first-in-class, cell-permeable small-molecule fluorescent probe engineered specifically for the high-contrast visualization of centrioles, cilia, and flagella without requiring genetic manipulation. Its highly straightforward “add and image” protocol allows for centrioles to be stained as fast as within 1h. It operates on a unique dual-ligand principle designed to exploit the specific structural architecture of these organelles: it physically couples two different microtubule-binding agents (cabazitaxel and a peloruside A analog) via a PEG linker, allowing the probe to simultaneously bind to both the inner and outer microtubule-binding sites that exist in close juxtaposition exclusively within the microtubule triplets and doublets of centriolar and axonemal structures (Figure 1). This bivalent binding mechanism confers CenSpark650 with an exceptional selectivity for centrioles over standard, indiscriminate cellular tubulin networks, and because it is fluorogenic, its far-red (670 nm) fluorescence intensity increases nearly ninefold only upon target binding, resulting in a highly specific, low-background signal ideal for live cell imaging.

• First-in-Class Centriole and Cilia Selective Small-Molecule Probe: CenSpark650 addresses a long-standing gap in cell biology by providing a generic, small-molecule probe specifically engineered for the quick and selective visualization of centrioles and cilia in live cells with minimal technical limitations.
• No Genetic Manipulation Required: It allows to monitor centrioles, cilia, and flagella effortlessly in living or fixed specimens without needing complex, time-consuming, or disruptive molecular genetic tools.
• Exquisite Specificity via Dual-Ligand Design: Utilizing a clever inside-outside "bridge" topology (coupling cabazitaxel and a peloruside A analog) via PEG linkers), the probe locks onto the unique juxtaposition of microtubule triplets and doublets found only in centrioles and cilia. This translates to an exceptional selectivity over standard, indiscriminate tubulin probes on the market.
• Fluorogenic: CenSpark650 features a large fluorescence intensity increase upon binding to its target. This fluorogenicity dramatically minimizes background and improves overall image contrast.
• Optimized Far-Red Imaging: Operating in the 650 nm range, the probe minimizes phototoxicity and auto-fluorescence in living samples while leaving open valuable channels for multiplexing with green or red fluorophores (e.g., GFP or RFP)
• Exceptional High-Resolution Performance: Thanks to the high superresolution compatibility of the SiR-fluorophore, the probe is fully compatible with live-cell STED super-resolution microscopy , allowing researchers to cleanly capture the fine 3D architecture of mature centrioles and newly forming procentrioles.
• Perfect for Long-Term Live Tracking: CenSpark650 possesses a very low dissociation rate, meaning it stays bound to its target structure for a very long time. It enables continuous tracking of centriolar movement over consecutive cell cycles for up to 2 full days from just a single, initial 1-hour staining.
• No toxicity: Long-term live imaging with CenSpark650 exhibits no cell toxicity and does not alter cell cycle progression, mitotic duration, viability, and microtubule dynamics.
• Fixed-Cell Compatibility: Beyond live tracking, CenSpark650 is versatile enough to be compatible with fixed cells (particularly with acetone or glutaraldehyde fixation workflows), offering seamless adaptation into existing lab protocols and full compatibility with standard IF.