Monte Carlo study of B-cell receptor clustering mediated by antigen crosslinking and directed transport

It is known from experiments that in the presence of soluble antigen,B-cell receptors(BCRs)assemble into microclusters and then collect into a macrocluster known as a‘cap’.However,the mechanisms of BCR cluster formation during recognition of soluble antigens remain unclear.In previous work,we demonstrated that effective intrinsic attractions among BCRs can lead to the formation of small microclusters of BCR molecules.The effective intrinsic attractions could be caused by multivalent antigen binding,association with lipid rafts,or other biochemical factors.In the present study,we have developed and studied a Monte Carlo model of BCR clustering mediated by explicit binding and crosslinking of soluble bivalent antigens.Antigen crosslinking is shown to microcluster BCRs in an affinity-dependent manner and also in a biologically relevant timescale;however,antigen crosslinking alone does not appear to be sufficient for the formation of a single macrocluster of receptor molecules.We show that directed transport of BCRs is needed to drive the formation of large macroclusters.We constructed a simple model of directed transport,where BCR molecules diffuse towards the largest cluster or towards a random BCR microcluster,which results in a single macrocluster of receptor molecules.The mechanisms for both types of directed transport are compared using network-based metrics.We also develop and use appropriate network measures to analyze the effect of BCR and antigen concentration on BCR clustering,the stability of the formed clusters over time and the size of BCR–antigen crosslinked chains.

Clustering of surface NMDA receptors is mainly mediated by the C-terminus of GluN2A in cultured rat hippocampal neurons

N-methyI-D-aspartate receptors （NMDARs） containing different GluN2 subunits play distinct roles in synaptic plasticity. Such differences may not only be determined by the channel properties, but also by differential surface distribution and synaptic localization. In the present study, using a Cy3-conjugated Fab fragment of the GFP antibody to label surface-located GluN2 subunits tagged with GFP at the N-terminus, we observed the membrane distribution patterns of GluN2A- or GluN2B-containing NMDARs in cultured rat hippocampal neurons. We found that surface NMDARs containing GluN2A, but not those containing GluN2B, were inclined to cluster at DIV7. Swapping the carboxyl termini of the GluN2 subunits completely reversed these distribution patterns. In addition, surface NMDARs containing GluN2A were preferentially associated with PSD-95. Taken together, the results of our study suggest that the clustering distribution of GluN2A- containing NMDARs is determined by the GluN2A C-terminus, and its interaction with PSD-95 plays an important role in this process.

LncRNA AFAP1-AS1/miR-27b-3p/VEGF-C axis modulates stemness characteristics in cervical cancer cells

Background:Long non-coding RNA(lncRNA)actin filament-associated protein 1 antisense RNA 1(AFAP1-AS1)functions as a competing endogenous RNA to regulate target genes expression by sponging microRNAs(miRs)to play cancer-promoting roles in cancer stem cells.However,the regulatory mechanism of AFAP1-AS1 in cervical cancer(CC)stem cells is unknown.The present study aimed to provide a new therapeutic target for the clinical treatment of CC.Methods:Hyaluronic acid receptor cluster of differentiation 44 variant exon 6(CD44v6)(+)CC cells were isolated by flow cytometry(FCM).Small interfering RNAs of AFAP1-AS1(siAFAP1-AS1)were transfected into the(CD44v6)(+)cells.The levels of AFAP1-AS1 were measured by quantitative real-time PCR(qRT-PCR).Sphere formation assay,cell cycle analysis,and Western blotting were used to detect the effect of siAFAP1-AS1.RNA pull-down and luciferase reporter assay were used to verify the relationship between miR-27b-3p and AFAP1-AS1 or vascular endothelial growth factor(VEGF)-C.Results:CD44v6(+)CCcells had remarkable stemness and a high level ofAFAP1-AS1.However,AFAP1-AS1knockdownwithsiAFAP1-AS1suppressed the cell cycle transitionofG(1)/S phase and inhibited self-renewal ofCD44v6(+)CCcells,the levels of the stemnessmarkers octamer-binding transcription factor 4(OCT4),osteopontin(OPN),and cluster of differentiation 133(CD133),and the epithelialmesenchymal transition(EMT)-related proteins Twist1,matrix metalloprotease(MMP)-9,and VEGF-C.In the mechanism study,miR-27b-3p/VEGF-C signaling was demonstrated to be a key downstream of AFAP1-AS1 in the CD44v6(+)CC cells.Conclusions:LncRNA AFAP1-AS1 knockdown inhibits the CC cell stemness by upregulating miR-27b-3p to suppress VEGF-C.