Mechanistiac Studies Of Traction Force And PKC? In B Cell Activation And Function

After encountering and recognizing antigen,B lymphocytes(B cells)response into immuno-activation,followed by proliferation,differentiation and antibody secretion,which eventually mediate humoral immunity.It is reported B cell undergoes a spreading-then-contraction responses upon the contact of antigen which are presented by planar lipid bilayer.These responses are induced by the traction force generated by the B cell.However,due to the lack of measurement methods suitable for B cells,it has not been quantitatively analyzed that the temporal and spatial dynamics of traction force during B cell activation,nor the physiological significance of traction force during this process yet.In this study,we improved traditional traction force microscopy technique to characterize the spatial-temporal dynamics of traction force generated during B cell activation.Using an artificial condition mimicking in-vivo microenvironments,we showed that B cell activation required the generation of centripetal traction force,which gradually grew with time,with a maximal value of 10-20 n N.Further experiments revealed that the recognition of specific antigen by B cell receptor(BCR)is necessary for traction force generation,and BCR also acts as the anchoring rivets and traction force–transmitter to antigen.B cell generates traction force to aggregate antigen into the central region of the cell-substrate contact surface via BCR,thus helps enhance the formation of immuno-synapse,and eventually promotes the activation efficiency of B cell.Mechanistically,the remodeling of intracellular actin and the sliding of myosin and dynein along the cytoskeleton altogether provide the contractility contributed to traction force generation.Moreover,membrane-proximal BCR signaling molecules and adaptor molecules linking BCR microclusters and motor proteins are also required for the generation of cellular traction forces.Isotype-switched B cells including memory B cells,expressing immunoglobulin G(Ig G)-BCRs generates greater traction forces than do mature na?ve B cells expressing Ig M-BCRs during activation,which is related to the longer cytoplasmic tail of membrane-bound Ig G(m Ig G).We found a positive correlation between the strength of the traction force and the level of B cell activation,which was indicated by mean fluorescence intensity of the BCR microclusters.Thus the measurement of traction force is potentially capable for characterizing the activation level of B cell.Furthermore,primary B cells from patients with rheumatoid arthritis generates greater traction forces than do B cells from healthy donors in response to antigen stimulation,which suggests the potential of the traction force microscopy technique improved in this study for convenient and rapid clinical diagnosis in the future.PKC?,a novel member of the protein kinase C family,is able to bind to filamentous actin and regulates the phosphorylation of myosin.However,the mechanism by which PKC? mediates B cell activation still remains unclear.In this study,we tested the primary cells of PKC? knockout transgenic mice with the improved traction microscopy.It was revealed that the loss of PKC? resulted in the impaired traction force generation during B cell activation.Thus we proposed that PKC? positively regulates B cell activation by affecting the traction force generation.Moreover,PKC? is also involved in the production of antibodies and the formation of germinal centers in vivo,which are known as key physiological functions of mature B cell after activation.In summary,in this study we established an improved traction force microscopy system suitable for B cells;found the general mechanisms for traction force generation during B cell activation and proposed its physiological significance;and using this system,verified and further discovered the mechanisms by which PKC? regulates B cell activation and functions in vivo.These results will help further reveal the regulatory signaling network of B cell activation and functions,as well as provide a theoretical basis for the diagnosis and treatment of related diseases.