Limits...
Spinophilin participates in information transfer at immunological synapses.

Bloom O, Unternaehrer JJ, Jiang A, Shin JS, Delamarre L, Allen P, Mellman I - J. Cell Biol. (2008)

Bottom Line: In DCs interacting with T cells, spinophilin is polarized dynamically to contact sites in an antigen-dependent manner.It is also required for optimal T cell activation because DCs derived from mice lacking spinophilin exhibit defects in antigen presentation both in vitro and in vivo.Thus, spinophilin may play analogous roles in information transfer at both neuronal and immunological synapses.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Ludwig Institute for Cancer Research, New Haven, CT 06520, USA.

ABSTRACT
The adaptive immune response is initiated by the presentation of peptides bound to major histocompatibility complex molecules on dendritic cells (DCs) to antigen-specific T lymphocytes at a junction termed the immunological synapse. Although much attention has been paid to cytoplasmic events on the T cell side of the synapse, little is known concerning events on the DC side. We have sought signal transduction components of the neuronal synapse that were also expressed by DCs. One such protein is spinophilin, a scaffolding protein of neuronal dendritic spines that regulates synaptic transmission. In inactive, immature DCs, spinophilin is located throughout the cytoplasm but redistributes to the plasma membrane upon stimulus-induced maturation. In DCs interacting with T cells, spinophilin is polarized dynamically to contact sites in an antigen-dependent manner. It is also required for optimal T cell activation because DCs derived from mice lacking spinophilin exhibit defects in antigen presentation both in vitro and in vivo. Thus, spinophilin may play analogous roles in information transfer at both neuronal and immunological synapses.

Show MeSH

Related in: MedlinePlus

Real-time imaging of spinophilin polarizing at the IS. (a) DCs expressing spinophilin-GFP that were cultured in the presence (+Ag; top, n = 6 conjugates) or absence (−Ag; middle, n = 3 conjugates) of agonist peptide together with antigen-specific CD4+ T cells that had been labeled red by the lipophilic PKH-26 dye (Sigma-Aldrich). In the presence of the antigen, spinophilin was polarized minutes after contact and remained polarized throughout the duration of the contact (30–60 min; a, top; and b). In the absence of antigen, spinophilin was distributed throughout the cytoplasm (a, middle; and b). As a control, the distribution of GFP alone in DCs contacting T cells (n = 3 conjugates) was imaged in the presence of the antigen and localized evenly throughout the cytoplasm (a, bottom). Time is indicated in minutes after imaging began and is rounded to the nearest minute. The bar in section a (middle) applies to the spinophilin-GFP +/− antigen. T, T cell. (b) The distribution of fluorescence signal for GFP alone with an antigen (gray) and spinophilin-GFP both with (green) and without an antigen (black) in live cells was analyzed at 0, 4, 14, and 20 min after imaging began (n = 9, 12, and 22 observations from 3, 3, and 6 conjugates, respectively, for GFP alone, spinophilin-GFP –Ag, and spinophilin-GFP +Ag). Spinophilin-GFP was most polarized toward the T cell in the presence of the antigen (P < 0.05 with respect to spinophilin-gfp –Ag; P < 0.002 with respect to GFP alone +Ag by Student's t test).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2315669&req=5

fig3: Real-time imaging of spinophilin polarizing at the IS. (a) DCs expressing spinophilin-GFP that were cultured in the presence (+Ag; top, n = 6 conjugates) or absence (−Ag; middle, n = 3 conjugates) of agonist peptide together with antigen-specific CD4+ T cells that had been labeled red by the lipophilic PKH-26 dye (Sigma-Aldrich). In the presence of the antigen, spinophilin was polarized minutes after contact and remained polarized throughout the duration of the contact (30–60 min; a, top; and b). In the absence of antigen, spinophilin was distributed throughout the cytoplasm (a, middle; and b). As a control, the distribution of GFP alone in DCs contacting T cells (n = 3 conjugates) was imaged in the presence of the antigen and localized evenly throughout the cytoplasm (a, bottom). Time is indicated in minutes after imaging began and is rounded to the nearest minute. The bar in section a (middle) applies to the spinophilin-GFP +/− antigen. T, T cell. (b) The distribution of fluorescence signal for GFP alone with an antigen (gray) and spinophilin-GFP both with (green) and without an antigen (black) in live cells was analyzed at 0, 4, 14, and 20 min after imaging began (n = 9, 12, and 22 observations from 3, 3, and 6 conjugates, respectively, for GFP alone, spinophilin-GFP –Ag, and spinophilin-GFP +Ag). Spinophilin-GFP was most polarized toward the T cell in the presence of the antigen (P < 0.05 with respect to spinophilin-gfp –Ag; P < 0.002 with respect to GFP alone +Ag by Student's t test).

Mentions: Within T cells, many proteins have been found to localize transiently to the contact site with APCs (Dustin et al., 2006). Far less is known, however, about the dynamics of protein trafficking to the IS from the APC side. To understand the relationship between contact duration and spinophilin polarization, we next used video confocal microscopy to examine the distribution of spinophilin fused to GFP or GFP alone in living DCs allowed to interact with antigen-specific T cells. Immature DCs were transduced with a recombinant retrovirus expressing spinophilin-GFP or GFP alone and maintained in culture. Mature DCs were then cultured in the presence (n = 6 conjugates) or absence (n = 3 conjugates) of an agonist peptide (moth cytochrome c, aa 88–103), adhered to coverslips, and cocultured with antigen-specific CD4+ T cells. In fixed cells, spinophilin-GFP was distributed similarly to the endogenous protein (detected by polyclonal antisera) and could be found polarized toward the T cell contact sites in the presence of an agonist peptide (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200711149/DC1). In live cells, this localization in conjugates with polarized spinophilin was even more dramatic, with spinophilin-GFP rapidly (<5 min) polarizing toward the T cell contact for the duration of the contact (Fig. 3 a, top; Fig. 3 b; and Videos 1 and 2). The polarization was barely evident in those few conjugates that formed in the absence of the antigen (Fig. 3 a, middle; Fig. 3 b; and Video 3). Free GFP, a cytosolic marker reporting overall cell shape, did not exhibit any detectable polarization in the presence of the antigen (n = 3 conjugates; Fig. 3 a, bottom; and Fig. 3 b). The distribution of GFP with the antigen or spinophilin-GFP with and without the antigen was quantified in a region of interest (ROI) both toward and away from the contact site at 0, 4, 14, and 20 min after imaging began. The ratio of fluorescence intensity in an ROI toward/away from the contact site was determined and averaged over time. As shown in Fig. 3 b, spinophilin-GFP with the antigen was 2.6-fold enriched at the contact site and 1.4-fold enriched without the antigen. There was no enrichment at the contact site of GFP alone with the antigen.


Spinophilin participates in information transfer at immunological synapses.

Bloom O, Unternaehrer JJ, Jiang A, Shin JS, Delamarre L, Allen P, Mellman I - J. Cell Biol. (2008)

Real-time imaging of spinophilin polarizing at the IS. (a) DCs expressing spinophilin-GFP that were cultured in the presence (+Ag; top, n = 6 conjugates) or absence (−Ag; middle, n = 3 conjugates) of agonist peptide together with antigen-specific CD4+ T cells that had been labeled red by the lipophilic PKH-26 dye (Sigma-Aldrich). In the presence of the antigen, spinophilin was polarized minutes after contact and remained polarized throughout the duration of the contact (30–60 min; a, top; and b). In the absence of antigen, spinophilin was distributed throughout the cytoplasm (a, middle; and b). As a control, the distribution of GFP alone in DCs contacting T cells (n = 3 conjugates) was imaged in the presence of the antigen and localized evenly throughout the cytoplasm (a, bottom). Time is indicated in minutes after imaging began and is rounded to the nearest minute. The bar in section a (middle) applies to the spinophilin-GFP +/− antigen. T, T cell. (b) The distribution of fluorescence signal for GFP alone with an antigen (gray) and spinophilin-GFP both with (green) and without an antigen (black) in live cells was analyzed at 0, 4, 14, and 20 min after imaging began (n = 9, 12, and 22 observations from 3, 3, and 6 conjugates, respectively, for GFP alone, spinophilin-GFP –Ag, and spinophilin-GFP +Ag). Spinophilin-GFP was most polarized toward the T cell in the presence of the antigen (P < 0.05 with respect to spinophilin-gfp –Ag; P < 0.002 with respect to GFP alone +Ag by Student's t test).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2315669&req=5

fig3: Real-time imaging of spinophilin polarizing at the IS. (a) DCs expressing spinophilin-GFP that were cultured in the presence (+Ag; top, n = 6 conjugates) or absence (−Ag; middle, n = 3 conjugates) of agonist peptide together with antigen-specific CD4+ T cells that had been labeled red by the lipophilic PKH-26 dye (Sigma-Aldrich). In the presence of the antigen, spinophilin was polarized minutes after contact and remained polarized throughout the duration of the contact (30–60 min; a, top; and b). In the absence of antigen, spinophilin was distributed throughout the cytoplasm (a, middle; and b). As a control, the distribution of GFP alone in DCs contacting T cells (n = 3 conjugates) was imaged in the presence of the antigen and localized evenly throughout the cytoplasm (a, bottom). Time is indicated in minutes after imaging began and is rounded to the nearest minute. The bar in section a (middle) applies to the spinophilin-GFP +/− antigen. T, T cell. (b) The distribution of fluorescence signal for GFP alone with an antigen (gray) and spinophilin-GFP both with (green) and without an antigen (black) in live cells was analyzed at 0, 4, 14, and 20 min after imaging began (n = 9, 12, and 22 observations from 3, 3, and 6 conjugates, respectively, for GFP alone, spinophilin-GFP –Ag, and spinophilin-GFP +Ag). Spinophilin-GFP was most polarized toward the T cell in the presence of the antigen (P < 0.05 with respect to spinophilin-gfp –Ag; P < 0.002 with respect to GFP alone +Ag by Student's t test).
Mentions: Within T cells, many proteins have been found to localize transiently to the contact site with APCs (Dustin et al., 2006). Far less is known, however, about the dynamics of protein trafficking to the IS from the APC side. To understand the relationship between contact duration and spinophilin polarization, we next used video confocal microscopy to examine the distribution of spinophilin fused to GFP or GFP alone in living DCs allowed to interact with antigen-specific T cells. Immature DCs were transduced with a recombinant retrovirus expressing spinophilin-GFP or GFP alone and maintained in culture. Mature DCs were then cultured in the presence (n = 6 conjugates) or absence (n = 3 conjugates) of an agonist peptide (moth cytochrome c, aa 88–103), adhered to coverslips, and cocultured with antigen-specific CD4+ T cells. In fixed cells, spinophilin-GFP was distributed similarly to the endogenous protein (detected by polyclonal antisera) and could be found polarized toward the T cell contact sites in the presence of an agonist peptide (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200711149/DC1). In live cells, this localization in conjugates with polarized spinophilin was even more dramatic, with spinophilin-GFP rapidly (<5 min) polarizing toward the T cell contact for the duration of the contact (Fig. 3 a, top; Fig. 3 b; and Videos 1 and 2). The polarization was barely evident in those few conjugates that formed in the absence of the antigen (Fig. 3 a, middle; Fig. 3 b; and Video 3). Free GFP, a cytosolic marker reporting overall cell shape, did not exhibit any detectable polarization in the presence of the antigen (n = 3 conjugates; Fig. 3 a, bottom; and Fig. 3 b). The distribution of GFP with the antigen or spinophilin-GFP with and without the antigen was quantified in a region of interest (ROI) both toward and away from the contact site at 0, 4, 14, and 20 min after imaging began. The ratio of fluorescence intensity in an ROI toward/away from the contact site was determined and averaged over time. As shown in Fig. 3 b, spinophilin-GFP with the antigen was 2.6-fold enriched at the contact site and 1.4-fold enriched without the antigen. There was no enrichment at the contact site of GFP alone with the antigen.

Bottom Line: In DCs interacting with T cells, spinophilin is polarized dynamically to contact sites in an antigen-dependent manner.It is also required for optimal T cell activation because DCs derived from mice lacking spinophilin exhibit defects in antigen presentation both in vitro and in vivo.Thus, spinophilin may play analogous roles in information transfer at both neuronal and immunological synapses.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Ludwig Institute for Cancer Research, New Haven, CT 06520, USA.

ABSTRACT
The adaptive immune response is initiated by the presentation of peptides bound to major histocompatibility complex molecules on dendritic cells (DCs) to antigen-specific T lymphocytes at a junction termed the immunological synapse. Although much attention has been paid to cytoplasmic events on the T cell side of the synapse, little is known concerning events on the DC side. We have sought signal transduction components of the neuronal synapse that were also expressed by DCs. One such protein is spinophilin, a scaffolding protein of neuronal dendritic spines that regulates synaptic transmission. In inactive, immature DCs, spinophilin is located throughout the cytoplasm but redistributes to the plasma membrane upon stimulus-induced maturation. In DCs interacting with T cells, spinophilin is polarized dynamically to contact sites in an antigen-dependent manner. It is also required for optimal T cell activation because DCs derived from mice lacking spinophilin exhibit defects in antigen presentation both in vitro and in vivo. Thus, spinophilin may play analogous roles in information transfer at both neuronal and immunological synapses.

Show MeSH
Related in: MedlinePlus