Chemical fixation creates nanoscale clusters on the cell surface by aggregating membrane proteins

Abstract

Chemical fixations have been thought to preserve the structures of the cells or tissues. However, given that the fixatives create crosslinks or aggregate proteins, there is a possibility that these fixatives create nanoscale artefacts by aggregation of membrane proteins which move around freely to some extent on the cell surface. Despite this, little research has been conducted about this problem, probably because there has been no method for observing cell surface structures at the nanoscale. In this study, we have developed a method to observe cell surfaces stably and with high resolution using atomic force microscopy and a microporous silicon nitride membrane. We demonstrate that the size of the protrusions on the cell surface is increased after treatment with three commonly used fixatives and show that these protrusions were created by the aggregation of membrane proteins by fixatives. These results call attention when observing fixed cell surfaces at the nanoscale.

Nano‐Infrared Imaging and Spectroscopy of Animal Cells in Liquid Environment

“…Paraformaldehyde is known to crosslink proteins and creates surface clusters of membrane proteins in mammalian cells. [45] The chemical composition and structure, and the localization and heterogeneity of sample biochemistry that is revealed in the nano-IR spectra reported here for the case of the cell membrane and cellular substructures near the membrane agrees well with that obtained from SERS experiments from the same cell line. [35] Nevertheless, the complementarity of both vibrational near-field approaches becomes obvious when we compare the relative contributions of modes from different molecular classes.…”

Nano‐Infrared Imaging and Spectroscopy of Animal Cells in Liquid Environment

“…Paraformaldehyde is known to crosslink proteins and creates surface clusters of membrane proteins in mammalian cells. [45] The chemical composition and structure, and the localization and heterogeneity of sample biochemistry that is revealed in the nano-IR spectra reported here for the case of the cell membrane and cellular substructures near the membrane agrees well with that obtained from SERS experiments from the same cell line. [35] Nevertheless, the complementarity of both vibrational near-field approaches becomes obvious when we compare the relative contributions of modes from different molecular classes.…”

Native spike flexibility revealed by BSL3 Cryo-ET of active SARS-CoV-2 virions

“…Figure 1–figure supplement 3c also shows that two main shapes, either spherical or ellipsoidal, were observed. As pointed out in previous studies 21,26 , inactivation has a direct effect on virion shape and flexibility, where native form shows higher polymorphism.…”

An aldehyde-crosslinking mitochondrial probe for STED imaging in fixed cells

“…As a fixable mitochondrial dye, PKMO FX offers STED images of fixed cells whose quality is approaching those acquired in live cells, with only minor background staining. We also recapitulate that GA fixation gives minimal structural distortion to the delicate ultrastructure of mitochondria compared to FA fixation ( 55 , 56 ). Moreover, optimized fixation protocol combining GA and FA can mostly preserve the membrane structure of mitochondria while at the same time enabling immunostainings.…”

Nano‐Infrared Imaging and Spectroscopy of Animal Cells in Liquid Environment

“…Paraformaldehyde is known to crosslink proteins and creates surface clusters of membrane proteins in mammalian cells. [45] The chemical composition and structure, and the localization and heterogeneity of sample biochemistry that is revealed in the nano-IR spectra reported here for the case of the cell membrane and cellular substructures near the membrane agrees well with that obtained from SERS experiments from the same cell line. [35] Nevertheless, the complementarity of both vibrational near-field approaches becomes obvious when we compare the relative contributions of modes from different molecular classes.…”

Native spike flexibility revealed by BSL3 Cryo-ET of active SARS-CoV-2 virions

“…Figure 1–figure supplement 3c also shows that two main shapes, either spherical or ellipsoidal, were observed. As pointed out in previous studies 21,26 , inactivation has a direct effect on virion shape and flexibility, where native form shows higher polymorphism.…”

An aldehyde-crosslinking mitochondrial probe for STED imaging in fixed cells

“…As a fixable mitochondrial dye, PKMO FX offers STED images of fixed cells whose quality is approaching those acquired in live cells, with only minor background staining. We also recapitulate that GA fixation gives minimal structural distortion to the delicate ultrastructure of mitochondria compared to FA fixation ( 55 , 56 ). Moreover, optimized fixation protocol combining GA and FA can mostly preserve the membrane structure of mitochondria while at the same time enabling immunostainings.…”

Nano‐Infrared Imaging and Spectroscopy of Animal Cells in Liquid Environment

“…Paraformaldehyde is known to crosslink proteins and creates surface clusters of membrane proteins in mammalian cells. [45] The chemical composition and structure, and the localization and heterogeneity of sample biochemistry that is revealed in the nano-IR spectra reported here for the case of the cell membrane and cellular substructures near the membrane agrees well with that obtained from SERS experiments from the same cell line. [35] Nevertheless, the complementarity of both vibrational near-field approaches becomes obvious when we compare the relative contributions of modes from different molecular classes.…”

Native spike flexibility revealed by BSL3 Cryo-ET of active SARS-CoV-2 virions

“…Figure 1–figure supplement 3c also shows that two main shapes, either spherical or ellipsoidal, were observed. As pointed out in previous studies 21,26 , inactivation has a direct effect on virion shape and flexibility, where native form shows higher polymorphism.…”

An aldehyde-crosslinking mitochondrial probe for STED imaging in fixed cells

“…As a fixable mitochondrial dye, PKMO FX offers STED images of fixed cells whose quality is approaching those acquired in live cells, with only minor background staining. We also recapitulate that GA fixation gives minimal structural distortion to the delicate ultrastructure of mitochondria compared to FA fixation ( 55 , 56 ). Moreover, optimized fixation protocol combining GA and FA can mostly preserve the membrane structure of mitochondria while at the same time enabling immunostainings.…”