Biology subjects

Frolikova, M.

Publications and source records attributed to Frolikova, M..

3 recordsLinked to original sources

bioRxiv2025

Spindle-localized F-actin regulates polar MTOC organization and the fidelity of meiotic spindle formation

Mammalian oocytes are notoriously prone to chromosome segregation errors leading to aneuploidy. The spindle provides the machinery for accurate chromosome segregation during cell division. Mammalian oocytes lack centrioles and, therefore, the meiotic spindle relies on the organization of numerous acentriolar microtubule organizing centers into two poles (polar MTOCs, pMTOCs). The traditional view is that, in mammalian oocytes, microtubules are the sole cytoskeletal component responsible for regulating pMTOC organization and spindle assembly. We identified a novel F-actin pool that surrounds pMTOCs, forming F-actin cage-like structure. We demonstrated that F-actin localization on the spindle depends on unconventional myosins X and VIIb. Selective disruption of spindle-localized F-actin, using myosin X/VIIb knockdown oocytes or photoswitchable Optojasp-1, perturbed pMTOC organization, leading to unfocused spindle poles and chromosome missegregation. Here, we unveil an important function of F-actin in regulating pMTOC organization, a critical process for ensuring the fidelity of meiotic spindle formation and proper chromosome segregation.

cell biology↗

bioRxiv2024

Protamine 2 Deficiency Results In Septin 12 Abnormalities

There is a well-established link between abnormal sperm chromatin states and poor motility, however, how these two processes are interdependent is unknown. Here, we identified a possible mechanistic insight by showing that Protamine 2, a nuclear DNA packaging protein in sperm, directly interacts with cytoskeletal protein Septin 12, which is associated with sperm motility. Septin 12 has several isoforms, and we show, that in the Prm2-/-sperm, the short one (Mw 36 kDa) is mislocalized, while two long isoforms (Mw 40 and 41 kDa) are unexpectedly lost in Prm2-/- sperm chromatin-bound protein fractions. Septin 12 co-immunoprecipitated with Protamine 2 in the testicular cell lysate of WT mice and with Lamin B1/B2/B3 in co-transfected HEK cells despite we did not observe changes in Lamin B2/B3 protein or SUN4 expression in Prm2-/-testes. Furthermore, the Prm2-/- sperm have on average a smaller sperm nucleus and aberrant acrosome biogenesis. In humans, patients with low sperm motility (asthenozoospermia) have imbalanced histone- protamine 1/2 ratio and modified levels of cytoskeletal proteins. We detected retained Septin 12 isoforms (Mw 40 and 41 kDa) in the sperm membrane, chromatin-bound and tubulin/mitochondria protein fractions, which was not true for healthy normozoospermic men. In conclusion, our findings expand the current knowledge regarding the connection between Protamine 2 and Septin 12 expression and localization, resulting in low sperm motility and morphological abnormalities.

cell biology↗

bioRxiv2023

A sample preparation procedure enables acquisition of 2-channel super-resolution 3D STED image of an entire oocyte

Super-resolution (SR) microscopy is a cutting-edge method that can provide detailed structural information with high resolution. However, the thickness of the specimen has been a major limitation for SR methods, and larger structures have posed a challenge. To overcome this, the key step is to optimize sample preparation to ensure optical homogeneity and clarity, which can enhance the capabilities of SR methods for the acquisition of thicker structures. Oocytes are the largest cells in the mammalian body and are crucial objects in reproductive biology. They are especially useful for studying membrane proteins. However, oocytes are extremely fragile and sensitive to mechanical manipulation and osmotic shocks, making sample preparation a critical and challenging step. We present an innovative, simple, and sensitive approach to oocyte sample preparation for 3D STED acquisition. This involves alcohol dehydration and mounting into a high refractive index medium. This extended preparation procedure allowed us to successfully obtain a unique 2-channel 3D STED super-resolution image of an entire mouse oocyte. By optimizing sample preparation, we can overcome the limitations of SR methods and obtain high-resolution images of larger structures, such as oocytes, Knowledge of which are important for understanding fundamental biological processes. RESEARCH HIGHLIGHTSO_LIThis study aimed to develop a successful sample preparation protocol for imaging mouse oocytes using 3D STED super-resolution microscopy. C_LIO_LIThe results showed that the oocyte sample was optically homogenous, enhancing the capability of the 3D STED method to capture high-resolution images throughout the full depth of the sample, resulting in highly similar SR images. C_LIO_LIThe 3D STED point spread function (PSF) pattern of the depletion laser was successfully secured throughout the entire volume of the sample, including the top and bottom. C_LIO_LIThis study presents the first-ever full volume 3D image of the mouse oocyte, which was acquired using a 2-channel 3D STED method. C_LI GRAPHICAL ABSTRACTIntroducing an extended sample preparation procedure resulted in an outstanding optical quality sample environment pronounced by high refractive index, high transparency, and minimal spherical aberration. This procedure allowed 3D STED within the entire oocyte. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

cell biology↗