Petitjean, I. I. | bioRxiv Science

bioRxiv2025

Anillin directly cross-links microtubules with filamentous actin

Complex morphogenetic processes such as cell division require a tight coordination of the activities of microtubules and actin filaments. There is evidence that anillin, conventionally known as an actin-binding and bundling protein, regulates microtubule-actin crosstalk during cell division. However, it is unknown whether anillin binds directly to microtubules and whether it is sufficient to establish crosslinking between microtubules and actin filaments. Here we address both questions by developing an in-vitro system to observe anillin-mediated interactions with actin filaments and dynamic microtubules using total internal reflection fluorescence microscopy. We find that anillin can interact directly with microtubules and promote microtubule bundling. We confirm that anillin binds and bundles actin filaments and find that it has a strong preference for actin bundles over individual filaments. Moreover, we show that anillin can directly crosslink microtubules and actin filaments, can cause sliding of actin filaments on the microtubule lattice, and can transport actin filaments by the growing microtubule tip. Our findings indicate that anillin can potentially serve as a direct regulator of microtubule-actin crosstalk, e.g. during cell division.

biophysics↗

bioRxiv2020

Physical properties and actin organization in embryonic stem cells depend on differentiation stage

The cellular cytoskeleton provides the cell with mechanical rigidity and mediates mechanical interaction between cells and with the extracellular environment. The actin structure plays a key role in regulating cellular behaviors like motility, cell sorting, or cell polarity. From the earliest stages of development, in naive stem cells, the critical mechanical role of the actin structure is becoming recognized as a vital cue for correct segregation and lineage control of cells and as a regulatory structure that controls several transcription factors. The ultrastructure of the earliest embryonic stem cells has not been investigated in living cells despite the fact that it is well-known that cells undergo morphological shape changes during the earliest stages of development. Here, we provide 3D investigations of the actin cytoskeleton of naive mouse embryonic stem cells (ESCs) in clusters of sizes relevant for early stage development using super resolution optical reconstruction microscopy (STORM). We quantitatively describe the morphological, cytoskeletal and mechanical changes appearing between cells in small clusters at the earliest stages of inner cell mass differentiation, as recapitulated by cells cultured under two media conditions, 2i and Serum/LIF, thus promoting the naive and first primed state, respectively. High resolution images of living stem cells showed that the peripheral actin structure undergoes a dramatic change between the two media conditions. The actin organization changed from being predominantly oriented parallel to the cell surface in 2i medium to a more radial orientation in Serum/LIF. Finally, using an optical trapping based technique, we detected micro-rheological differences in the cell periphery between the cells cultured in these two media, with results correlating well with the observed nano-architecture of the ESCs in the two different differentiation stages. These results pave the way for linking physical properties and cytoskeletal architecture to the development from naive stem cells to specialized cells. Statement of SignificanceCells receive mechanical signals and must provide mechanical feedback, therefore, physical properties are instrumental for cell-cell interactions. Mechanical signals mediated through the cell surface can significantly affect transport of signaling molecules and can influence biological processes like transcriptional regulation. To achieve a deeper insight into how the cytoskeletal structure is responsible for cell shape and material properties at the earliest stages of development, we employ super-resolution microscopy to image actin fibers in clusters of embryonic stem cells mimicking early development. By modification of the culturing conditions, we investigate how the actin cytoskeleton and micro-rheological properties of ESCs change between the naive ground state and the stage primed towards epiblast, thus revealing a correlation between differentiation stage and cytoskeletal structure.

biophysics↗