A highly stable RNA aptamer probe for the retinoblastoma protein in live cells.

Although RNA aptamers can show comparable or better specificity and affinity to antibodies and have the advantage of being able to access different live cell compartments, they are often much less stable . We report here the first aptamer that binds human retinoblastoma protein (RB) and is stable in live cells. RB is both a key protein in cell cycle control and also a tumour suppressor.

B cells rapidly target antigen and surface-derived MHCII into peripheral degradative compartments.

In order to mount high-affinity antibody responses, B cells internalise specific antigens and process them into peptides loaded onto MHCII for presentation to T helper cells (T cells). While the biochemical principles of antigen processing and MHCII loading have been well dissected, how the endosomal vesicle system is wired to enable these specific functions remains much less studied. Here, we performed a systematic microscopy-based analysis of antigen trafficking in B cells to reveal its route to the MHCII peptide-loading compartment (MIIC).

Dynamic reorganisation of intermediate filaments coordinates early B-cell activation.

During B-cell activation, the dynamic reorganisation of the cytoskeleton is crucial for multiple cellular responses, such as receptor signalling, cell spreading, antigen internalisation, intracellular trafficking, and antigen presentation. However, the role of intermediate filaments (IFs), which represent a major component of the mammalian cytoskeleton, is not well defined. Here, by using multiple super-resolution microscopy techniques, including direct stochastic optical reconstruction microscopy, we show that IFs in B cells undergo drastic reorganisation immediately upon antigen stimulation and that this reorganisation requires actin and microtubules.

The Lack of WIP Binding to Actin Results in Impaired B Cell Migration and Altered Humoral Immune Responses.

Wiskott-Aldrich syndrome protein (WASp) is a main cytoskeletal regulator in B cells. WASp-interacting protein (WIP) binds to and stabilizes WASp but also interacts with actin. Using mice with a mutated actin binding domain of WIP (WIPΔABD), we here investigated the role of WIP binding to actin during B cell activation.

Protein Kinase C-β Dictates B Cell Fate by Regulating Mitochondrial Remodeling, Metabolic Reprogramming, and Heme Biosynthesis.

PKCβ-null (Prkcb) mice are severely immunodeficient. Here we show that mice whose B cells lack PKCβ failed to form germinal centers and plasma cells, which undermined affinity maturation and antibody production in response to immunization. Moreover, these mice failed to develop plasma cells in response to viral infection.

Tuning of in vivo cognate B-T cell interactions by Intersectin 2 is required for effective anti-viral B cell immunity.

Wiskott-Aldrich syndrome (WAS) is an immune pathology associated with mutations in WAS protein (WASp) or in WASp interacting protein (WIP). Together with the small GTPase Cdc42 and other effectors, these proteins participate in the remodelling of the actin network downstream of BCR engagement. Here we show that mice lacking the adaptor protein ITSN2, a G-nucleotide exchange factor (GEF) for Cdc42 that also interacts with WASp and WIP, exhibited increased mortality during primary infection, incomplete protection after Flu vaccination, reduced germinal centre formation and impaired antibody responses to vaccination.

Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells.

B cells constitute an essential line of defense from pathogenic infections through the generation of class-switched antibody-secreting cells (ASCs) in germinal centers. Although this process is known to be regulated by follicular helper T (TfH) cells, the mechanism by which B cells initially seed germinal center reactions remains elusive. We found that NKT cells, a population of innate-like T lymphocytes, are critical for the induction of B cell immunity upon viral infection.

A switch from canonical to noncanonical autophagy shapes B cell responses.

Autophagy is important in a variety of cellular and pathophysiological situations; however, its role in immune responses remains elusive. Here, we show that among B cells, germinal center (GC) cells exhibited the highest rate of autophagy during viral infection. In contrast to mechanistic target of rapamycin complex 1-dependent canonical autophagy, GC B cell autophagy occurred predominantly through a noncanonical pathway.

Cdc42EP3/BORG2 and Septin Network Enables Mechano-transduction and the Emergence of Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) are non-cancerous cells found in solid tumors that remodel the tumor matrix and promote cancer invasion and angiogenesis. Here, we demonstrate that Cdc42EP3/BORG2 is required for the matrix remodeling, invasion, angiogenesis, and tumor-growth-promoting abilities of CAFs. Cdc42EP3 functions by coordinating the actin and septin networks.

Nanoscale organization and dynamics of the siglec CD22 cooperate with the cytoskeleton in restraining BCR signalling.

Receptor organization and dynamics at the cell membrane are important factors of signal transduction regulation. Using super-resolution microscopy and single-particle tracking, we show how the negative coreceptor CD22 works with the cortical cytoskeleton in restraining BCR signalling. In naïve B cells, we found endogenous CD22 to be highly mobile and organized into nanodomains.

Wiskott-Aldrich Syndrome Interacting Protein Deficiency Uncovers the Role of the Co-receptor CD19 as a Generic Hub for PI3 Kinase Signaling in B Cells.

Humans with Wiskott-Aldrich syndrome display a progressive immunological disorder associated with compromised Wiskott-Aldrich Syndrome Interacting Protein (WIP) function. Mice deficient in WIP recapitulate such an immunodeficiency that has been attributed to T cell dysfunction; however, any contribution of B cells is as yet undefined. Here we have shown that WIP deficiency resulted in defects in B cell homing, chemotaxis, survival, and differentiation, ultimately leading to diminished germinal center formation and antibody production.

Host response. Inflammation-induced disruption of SCS macrophages impairs B cell responses to secondary infection.

The layer of macrophages at the subcapsular sinus (SCS) captures pathogens entering the lymph node, preventing their global dissemination and triggering an immune response. However, how infection affects SCS macrophages remains largely unexplored. Here we show that infection and inflammation disrupt the organization of SCS macrophages in a manner that involves the migration of mature dendritic cells to the lymph node.

Cdc42 is a key regulator of B cell differentiation and is required for antiviral humoral immunity.

The small Rho GTPase Cdc42, known to interact with Wiskott-Aldrich syndrome (WAS) protein, is an important regulator of actin remodeling. Here, we show that genetic ablation of Cdc42 exclusively in the B cell lineage is sufficient to render mice unable to mount antibody responses. Indeed Cdc42-deficient mice are incapable of forming germinal centers or generating plasma B cells upon either viral infection or immunization.

STRIPAK components determine mode of cancer cell migration and metastasis.

The contractile actomyosin cytoskeleton and its connection to the plasma membrane are critical for control of cell shape and migration. We identify three STRIPAK complex components, FAM40A, FAM40B and STRN3, as regulators of the actomyosin cortex. We show that FAM40A negatively regulates the MST3 and MST4 kinases, which promote the co-localization of the contractile actomyosin machinery with the Ezrin/Radixin/Moesin family proteins by phosphorylating the inhibitors of PPP1CB, PPP1R14A-D.

Dynamics and stoichiometry of a regulated enhancer-binding protein in live Escherichia coli cells.

Bacterial enhancer-dependent transcription systems support major adaptive responses and offer a singular paradigm in gene control analogous to complex eukaryotic systems. Here we report new mechanistic insights into the control of one-membrane stress-responsive bacterial enhancer-dependent system. Using millisecond single-molecule fluorescence microscopy of live cells we determine the localizations, two-dimensional diffusion dynamics and stoichiometries of complexes of the bacterial enhancer-binding ATPase PspF during its action at promoters as regulated by inner membrane interacting negative controller PspA.

The actin and tetraspanin networks organize receptor nanoclusters to regulate B cell receptor-mediated signaling.

A key role is emerging for the cytoskeleton in coordinating receptor signaling, although the underlying molecular requirements remain unclear. Here we show that cytoskeleton disruption triggered signaling requiring not only the B cell receptor (BCR), but also the coreceptor CD19 and tetraspanin CD81, thus providing a mechanism for signal amplification upon surface-bound antigen stimulation. By using superresolution microscopy, we demonstrated that endogenous IgM, IgD, and CD19 exhibited distinct nanoscale organization within the plasma membrane of primary B cells.

B cell receptor-mediated antigen gathering requires ubiquitin ligase Cbl and adaptors Grb2 and Dok-3 to recruit dynein to the signaling microcluster.

The B cell receptor (BCR) mediates B cell antigen gathering and acquisition for presentation to T cells. Although the amount of antigen presentation to T cells determines the extent of B cell activation, the molecular mechanisms underlying antigen gathering remain unexplored. Here, through a combination of high-resolution imaging, genetics and quantitative mass spectrometry, we demonstrate that adaptors Grb2 and Dok-3, and ubiquitin ligase Cbl in signaling BCR microclusters mediate association with the microtubule motor dynein.

Dynamic cortical actin remodeling by ERM proteins controls BCR microcluster organization and integrity.

Signaling microclusters are a common feature of lymphocyte activation. However, the mechanisms controlling the size and organization of these discrete structures are poorly understood. The Ezrin-Radixin-Moesin (ERM) proteins, which link plasma membrane proteins with the actin cytoskeleton and regulate the steady-state diffusion dynamics of the B cell receptor (BCR), are transiently dephosphorylated upon antigen receptor stimulation.

Selective diffusion barriers separate membrane compartments.

We have investigated exchange of molecules between different membrane domains on a highly compartmentalized cell, the spermatozoon. Using Alexa Fluor 555-cholera toxin B-subunit we have observed clustering of preexisting GM1 gangliosides which diffused across the anterior acrosome-equatorial segment interface but did not access the postacrosome. By contrast, single lipid and protein molecules readily exchanged between all three domains, although they diffused more slowly on nearing and crossing to the postacrosome.

CD169(+) macrophages present lipid antigens to mediate early activation of iNKT cells in lymph nodes.

Invariant natural killer T cells (iNKT cells) are involved in the host defense against microbial infection. Although it is known that iNKT cells recognize glycolipids presented by CD1d, how and where they encounter antigen in vivo remains unclear. Here we used multiphoton microscopy to visualize the dynamics and activation of iNKT cells in lymph nodes.

The membrane skeleton controls diffusion dynamics and signaling through the B cell receptor.

Early events of B cell activation after B cell receptor (BCR) triggering have been well characterized. However, little is known about the steady state of the BCR on the cell surface. Here, we simultaneously visualize single BCR particles and components of the membrane skeleton.

Tracking diffusion of GM1 gangliosides and zona pellucida binding molecules in sperm plasma membranes following cholesterol efflux.

The molecules on mammalian spermatozoa that mediate recognition and binding to the zona pellucida of the egg are still not understood. Current concepts favour their assembly into multimolecular complexes in the plasma membrane in response to cholesterol efflux, an important step during sperm capacitation. Here, we track in real time diffusion of cross-linked clusters containing zona-binding molecules and GM1 gangliosides in the plasma membrane of live boar spermatozoa before and after cholesterol reduction.

Bayesian inference for improved single molecule fluorescence tracking.

Single molecule tracking is widely used to monitor the change in position of lipids and proteins in living cells. In many experiments in which molecules are tagged with a single or small number of fluorophores, the signal/noise ratio may be limiting, the number of molecules is not known, and fluorophore blinking and photobleaching can occur. All these factors make accurate tracking over long trajectories difficult and hence there is still a pressing need to develop better algorithms to extract the maximum information from a sequence of fluorescence images.

Nanopipette delivery of individual molecules to cellular compartments for single-molecule fluorescence tracking.

We have developed a new method, using a nanopipette, for controlled voltage-driven delivery of individual fluorescently labeled probe molecules to the plasma membrane which we used for single-molecule fluorescence tracking (SMT). The advantages of the method are 1), application of the probe to predefined regions on the membrane; 2), release of only one or a few molecules onto the cell surface; 3), when combined with total internal reflection fluorescence microscopy, very low background due to unbound molecules; and 4), the ability to first optimize the experiment and then repeat it on the same cell. We validated the method by performing an SMT study of the diffusion of individual membrane glycoproteins labeled with Atto 647-wheat germ agglutin in different surface domains of boar spermatozoa.