Centrioles Under the Microscope: Subcellular Insights

Centrioles play a crucial role in organizing microtubules and facilitating cell division. Their structured yet dynamic nature makes them key to cellular architecture, influencing mitotic spindle formation and cilia assembly. Despite their small size, advancements in imaging techniques have provided unprecedented insights into their organization and function.

Understanding centrioles at a subcellular level requires precise visualization methods capable of capturing their fine details. Various microscopy approaches allow researchers to examine their structure, arrangement, and behavior throughout the cell cycle.

Subcellular Placement And Arrangement

Centrioles reside within the centrosome, the primary microtubule-organizing center (MTOC) in animal cells. Typically, a pair—mother and daughter centrioles—are positioned orthogonally within a proteinaceous matrix known as the pericentriolar material (PCM). This arrangement influences microtubule nucleation and spindle orientation during cell division. The mother centriole, distinguished by distal and subdistal appendages, plays a dominant role in microtubule anchoring, while the daughter centriole matures over successive cycles.

Their positioning within the centrosome is tightly regulated by molecular interactions ensuring proper function. Proteins such as pericentrin and γ-tubulin contribute to PCM integrity, facilitating microtubule-nucleating complex recruitment. Centriole engagement—a tethering between mother and daughter centrioles—prevents premature duplication, ensuring replication occurs only once per cycle. Disruptions in this arrangement, seen in certain cancers and ciliopathies, can lead to aberrant centrosome amplification, chromosomal instability, and defective division.

Beyond mitosis, centrioles exhibit distinct spatial arrangements in differentiated cells. In multiciliated epithelial cells, hundreds of centrioles organize to support cilia formation, orchestrated by centriolar satellites enriched with PCM1 and CEP290. In neurons, centrioles are often asymmetrically positioned, influencing axon specification and intracellular transport. These variations underscore their adaptability to different cellular contexts.

Microscopy Approaches For Examination

Due to their small size—around 250 nm in diameter and 400–500 nm in length—traditional light microscopy lacks the resolution to capture centriole details. Advanced microscopy methods provide high-precision visualization.

Electron Microscopy

Electron microscopy (EM) provides high-resolution images, revealing centrioles’ ninefold radial symmetry and microtubule triplet organization. Transmission electron microscopy (TEM) is useful for examining cross-sections, while scanning electron microscopy (SEM) offers three-dimensional surface views. Cryo-electron tomography (cryo-ET) further refines our understanding by preserving centrioles in near-physiological conditions. A 2021 Nature Communications study used cryo-ET to visualize centriole-associated proteins, providing insights into stability and duplication. Despite its resolution, EM requires extensive sample preparation, which can introduce artifacts and limit live-cell imaging.

Fluorescence Microscopy

Fluorescence microscopy enables live-cell visualization of centriole behavior. By tagging centriole-associated proteins with fluorescent markers like GFP, researchers can track duplication, movement, and maturation. Confocal microscopy improves resolution by eliminating out-of-focus light, while structured illumination microscopy (SIM) enhances resolution beyond the diffraction limit. A 2020 Journal of Cell Biology study used SIM to analyze centriole elongation dynamics, revealing previously unrecognized structural transitions. While fluorescence microscopy provides valuable temporal information, its resolution is lower than electron microscopy, limiting detailed structural analysis.

Super-Resolution Techniques

Super-resolution microscopy techniques such as stimulated emission depletion (STED) and stochastic optical reconstruction microscopy (STORM) achieve resolutions below 50 nm, bridging the gap between fluorescence and electron microscopy. These methods allow visualization of centriole substructures, including the cartwheel—a key scaffold for assembly—at near-molecular resolution. STED microscopy has mapped proteins like SAS-6 and CEP135, critical for centriole formation. A 2022 Science Advances study employed STORM to investigate centriole-associated tubulin modifications, shedding light on their role in microtubule stability. While these techniques provide unprecedented detail, they require specialized instrumentation and fluorophores, making them less accessible for routine imaging. Prolonged exposure to high-intensity laser illumination can also lead to photobleaching, limiting long-term live-cell studies.

Structural Components Revealed

Centrioles feature a highly conserved cylindrical arrangement of microtubules, forming a ninefold symmetrical structure that serves as a scaffold for accessory proteins. At the core is a cartwheel-like structure composed of SAS-6 protein oligomers, establishing radial symmetry for proper assembly. Disruptions in SAS-6 function have been linked to defective duplication, leading to centrosomal abnormalities in developmental disorders.

Triplet microtubules surrounding the cartwheel are stabilized by proteins such as CEP135 and POC1, which reinforce rigidity and regulate elongation. The mature mother centriole’s distal and subdistal appendages, composed of proteins like CEP164 and ODF2, anchor microtubules and facilitate interactions with other cellular structures. These appendages are particularly significant in specialized cells where centrioles transition into basal bodies, supporting ciliary and flagellar function.

Beyond structural roles, centriolar proteins contribute to its dynamic nature, allowing adaptation to different cellular contexts. The PCM surrounding the centriole provides a platform for microtubule nucleation by recruiting γ-tubulin ring complexes, regulated by pericentrin and CDK5RAP2. The PCM undergoes remodeling in response to cellular signals, coordinating centriole activity with broader cellular functions.

Observations During Cell Cycle Progression

Centrioles undergo a tightly regulated duplication cycle to ensure each daughter cell inherits precisely two centrosomes, maintaining genomic stability. This process begins in late G1 when existing centrioles initiate new procentrioles. Polo-like kinase 4 (PLK4) triggers this process by recruiting SAS-6 and STIL to form the cartwheel scaffold. As the cell progresses into S phase, procentrioles elongate alongside the original centrioles.

During G2, centrioles mature, incorporating additional proteins for stability and functionality. The mother centrioles develop fully formed appendages, preparing for spindle organization. This maturation is facilitated by CEP295, ensuring procentrioles attain competence for independent duplication. Just before mitosis, centrosomes separate and migrate to opposite poles, guided by motor proteins like dynein and kinesin. Proper positioning is crucial for spindle orientation, as centrioles direct chromosome segregation.

Formation Of Basal Bodies

Centrioles serve as templates for basal body formation, essential for cilia and flagella development. A mature mother centriole migrates to the cell cortex, undergoing modifications to template axonemal microtubules. The transition to basal body is tightly regulated, ensuring proper ciliary assembly. Distal appendages anchor basal bodies to the plasma membrane, facilitating axoneme emergence. This process is particularly evident in multiciliated cells, where hundreds of basal bodies support coordinated ciliary beating, critical for mucus clearance and cerebrospinal fluid circulation.

Proteins such as CEP164 and Chibby regulate membrane docking, while centrin maintains basal body stability. Once anchored, basal bodies recruit intraflagellar transport (IFT) proteins, mediating ciliary component delivery. Disruptions in basal body formation can result in ciliopathies, disorders characterized by defective ciliary function. For example, mutations in CEP290 are implicated in Joubert syndrome, marked by neurological abnormalities due to impaired ciliary signaling. Studying basal body formation provides insights into how centrioles contribute to specialized cellular structures and their broader significance in developmental biology and disease.