Visualizing intra- and extracellular vesicles of B cells using expansion microscopy
Tarczewska, Maria (2023-05-01)
Visualizing intra- and extracellular vesicles of B cells using expansion microscopy
Tarczewska, Maria
(01.05.2023)
Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.
avoin
Julkaisun pysyvä osoite on:
https://urn.fi/URN:NBN:fi-fe2023053050186
https://urn.fi/URN:NBN:fi-fe2023053050186
Tiivistelmä
B lymphocytes (B cells) are a part of the adaptive immune system, where they are responsible for the production of antibodies against specific antigens. Upon antigen encounter, they become activated which alters their shape, size, and cytoskeleton. B cells release extracellular vesicles (EVs) that may help in antigen processing during activation. However, research data on the functions and characteristics of B cell EVs are scarce, and the content and function of these vesicles are largely unknown.
During B cell activation, vesicular populations such as lysosomes and endosomes traffic to the immune synapse where they have reported functions in antigen extraction and processing. Numerous proteins are found at the immune synapse and the role of many of them is unknown. Tetraspanins are known to play diverse roles in B cells. Here, the localization of tetraspanins CD63 and CD81 upon B cell activation was investigated using immunofluorescence microscopy (IF) and expansion microscopy (ExM). CD63 was shown to relocate to the perinuclear area of the Raji D1.3 cells upon B cell activation. CD81 was observed predominately in the cytoplasm and no changes in CD81 localization were observed.
The second part of the thesis focuses on imaging EVs using ExM. Studying vesicles is challenging due to their nanoscopic size. Here, ExM was implemented to study B-cell EVs. We show that ExM with in-gel suspended EVs allows for super-resolution imaging, although further data is required to verify the method's reliability. In the future, it can be used to study the morphology and content of EVs.
During B cell activation, vesicular populations such as lysosomes and endosomes traffic to the immune synapse where they have reported functions in antigen extraction and processing. Numerous proteins are found at the immune synapse and the role of many of them is unknown. Tetraspanins are known to play diverse roles in B cells. Here, the localization of tetraspanins CD63 and CD81 upon B cell activation was investigated using immunofluorescence microscopy (IF) and expansion microscopy (ExM). CD63 was shown to relocate to the perinuclear area of the Raji D1.3 cells upon B cell activation. CD81 was observed predominately in the cytoplasm and no changes in CD81 localization were observed.
The second part of the thesis focuses on imaging EVs using ExM. Studying vesicles is challenging due to their nanoscopic size. Here, ExM was implemented to study B-cell EVs. We show that ExM with in-gel suspended EVs allows for super-resolution imaging, although further data is required to verify the method's reliability. In the future, it can be used to study the morphology and content of EVs.