Biology subjects

Mantyla, E.

Publications and source records attributed to Mantyla, E..

3 recordsLinked to original sources

bioRxiv2022

Iterative immunostaining combined with expansion microscopy and image processing reveals nanoscopic network organization of nuclear lamina

Investigation of nuclear lamina architecture relies on super-resolved microscopy. However, epitope accessibility, labeling density, and detection precision of individual molecules pose challenges within the molecularly crowded nucleus. We developed iterative indirect immunofluorescence (IT-IF) staining approach combined with expansion microscopy (ExM) and structured illumination microscopy to improve super-resolution microscopy of subnuclear nanostructures like lamins. We prove that ExM is applicable in analyzing highly compacted nuclear multiprotein complexes such as viral capsids and provide technical improvements to ExM method including 3D-printed gel casting equipment. We show that in comparison to conventional immunostaining, IT-IF results in a higher signal-to-background -ratio and a mean fluorescence intensity by improving the labeling density. Moreover, we present a signal processing pipeline for noise estimation, denoising, and deblurring to aid in quantitative image analyses and provide this platform for the microscopy imaging community. Finally, we show the potential of signal-resolved IT-IF in quantitative super-resolution ExM imaging of nuclear lamina and reveal nanoscopic details of the lamin network organization - a prerequisite for studying intranuclear structural co-regulation of cell function and fate. (Words: 175)

molecular biology↗

bioRxiv2022

Nuclear lamina strain states revealed by intermolecular force biosensor

Nuclear lamins have been considered to be an important structural element of the nucleus. The nuclear lamina is thought both to shield DNA from excessive mechanical forces and to transmit mechanical forces onto the DNA. However, to date there is not yet a technical approach to directly measure mechanical forces on nuclear lamins at the protein level. To overcome this limitation, we developed a nanobody-based intermolecular tension FRET biosensor capable of measuring the mechanical strain of lamin filaments. Using this sensor, we were able to show that the nuclear lamina is subjected to significant force. These forces are dependent on nuclear volume, actomyosin contractility, functional LINC complex, chromatin condensation state, cell cycle, and EMT. Interestingly, large forces were also present on nucleoplasmic lamins, indicating that these lamins may also have an important mechanical role in the nucleus. Overall, we demonstrate that nanobody-based approach allows construction of novel force biosensors for mechanobiology studies.

biophysics↗

bioRxiv2021

Following the track: accuracy and reproducibility of predation assessment on artificial caterpillars

Experimental studies of biotic interactions in real field conditions are essential to understand the structure and functioning of ecological networks. The use of artificial caterpillars to mimic actual prey availability is generally seen as a standard approach to compare the activity and diversity of predators along environmental gradients. Yet, even with standardized material and procedures, biases may still affect data from multiple observers with different expertise. We used pictures of artificial caterpillars with or without various predation marks, in an online survey that was targeted for the participants of the project, to evaluate the reliability of predation marks identification made by non-scientists and by scientists with and without previous experience in predation mark identification. Pictures of artificial caterpillars displayed typical marks left by birds, mammals and arthropods, as well as non-predation marks ( false positive). 357 respondents scanned 7140 pictures of these pictures. Self-declared scientists were more confident and accurate in their observations than non-scientists, but the differences in correct identifications among scientists and non-scientists were low. Self-declared scientists with previous experience were also more accurate than scientists without previous experience, while there were no differences in self-confidence among scientists with and without previous experience. Accuracy in predation mark identification did not differ among types of predators, but respondents were more keen to identify marks left by birds or mammals than arthropods. Our results have practical implications for the design of multi-observer projects relying on artificial caterpillars as a proxy to assess predation intensity, in particular in the context of citizen science.

ecology↗