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Intercellular transfer to signalling endosomes regulates an ex vivo bone marrow niche.

Gillette JM, Larochelle A, Dunbar CE, Lippincott-Schwartz J - Nat. Cell Biol. (2009)

Bottom Line: Here, we use live-cell imaging to characterize both the site of contact between osteoblasts and haematopoietic progenitor cells (HPCs) and events at this site that result in downstream signalling responses important for niche maintenance.This caused osteoblasts to downregulate Smad signalling and increase production of stromal-derived factor-1 (SDF-1), a chemokine responsible for HSPC homing to bone marrow.These findings identify a mechanism involving intercellular transfer to signalling endosomes for targeted regulation of signalling and remodelling events within an ex vivo osteoblastic niche.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Program, National Institute of Child Health and Human Development, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892, USA.

ABSTRACT
Haematopoietic stem-progenitor cells (HSPCs) reside in the bone marrow niche, where interactions with osteoblasts provide essential cues for their proliferation and survival. Here, we use live-cell imaging to characterize both the site of contact between osteoblasts and haematopoietic progenitor cells (HPCs) and events at this site that result in downstream signalling responses important for niche maintenance. HPCs made prolonged contact with the osteoblast surface through a specialized membrane domain enriched in prominin 1, CD63 and rhodamine PE. At the contact site, portions of the specialized domain containing these molecules were taken up by the osteoblast and internalized into SARA-positive signalling endosomes. This caused osteoblasts to downregulate Smad signalling and increase production of stromal-derived factor-1 (SDF-1), a chemokine responsible for HSPC homing to bone marrow. These findings identify a mechanism involving intercellular transfer to signalling endosomes for targeted regulation of signalling and remodelling events within an ex vivo osteoblastic niche.

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Intercellular transfer correlates with decreased Smad 2/3 activation and an increased production of SDF-1 by the osteoblast. (a) N-Rh-PE (red) labeled KG1a cells were co-cultured for one hour with osteoblasts, which were then fixed and immunostained for SARA (green). Transferred N-Rh-PE could be detected within SARA positive vesicles in the osteoblast as indicated by the white circles. (b) Histogram of weighted co-localization coefficients for N-Rh-PE with a SARA positive endosome (n = 33 cells) and for SARA with Rab 7-GFP positive endosomes (n = 9 cells) indicates that transferred N-Rh-PE is detected within SARA positive endosomes, which are distinct from a Rab 7-GFP compartment. (c) Osteoblasts fixed and stained for Smad2/3 (green) have a high level of nuclear expression indicating activate Smad2/3. Following a transfer event, osteoblasts with detectable transfer (d) have a reduced nuclear localization of Smad2/3. The nuclear to cytoplasmic ratios of Smad2/3 signal intensity is quantified in (e). n > 50 osteoblasts +/- transfer. (f) Quantification of the percent of SDF-1 expressing osteoblasts without co-culture, following 1 h of co-culture, and 5 h of co-culture. (n > 100 osteoblasts or +/- transfer scored for 3 independent experiments). (g) SDF-1 immunofluorescence of a transfer positive osteoblast. White lines outline the osteoblasts in the field of view. (h) Osteoblasts were co-cultured with N-Rh-PE labeled KG1a cells for 1 h and then washed to remove all KG1a cells. SDF-1 expression was detected by immunofluorescence immediately following KG1a cell removal and then 4 h following KG1a cell removal. (n > 100 transfer positive osteoblasts for 3 independent experiments). (i) To evaluate whether transfer rather than KG1a cell contact mediated the increase in SDF-1 expression, KG1a/osteoblast co-cultures were treated with 10 mM methyl β cyclodextrin (MβCD), 2 μm cytocholasin D (Cyto D), or 80 μm Dynasore to allow for KG1a cell contact with osteoblasts, but not transfer. Drug treatment, which reduced N-Rh-PE transfer (Fig. 3h), but allowed for cell contact, resulted in a decreased percent of SDF-1 expressing osteoblasts when compared to control co-cultures. (n > 600 osteoblasts for 3 independent experiments)
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Figure 5: Intercellular transfer correlates with decreased Smad 2/3 activation and an increased production of SDF-1 by the osteoblast. (a) N-Rh-PE (red) labeled KG1a cells were co-cultured for one hour with osteoblasts, which were then fixed and immunostained for SARA (green). Transferred N-Rh-PE could be detected within SARA positive vesicles in the osteoblast as indicated by the white circles. (b) Histogram of weighted co-localization coefficients for N-Rh-PE with a SARA positive endosome (n = 33 cells) and for SARA with Rab 7-GFP positive endosomes (n = 9 cells) indicates that transferred N-Rh-PE is detected within SARA positive endosomes, which are distinct from a Rab 7-GFP compartment. (c) Osteoblasts fixed and stained for Smad2/3 (green) have a high level of nuclear expression indicating activate Smad2/3. Following a transfer event, osteoblasts with detectable transfer (d) have a reduced nuclear localization of Smad2/3. The nuclear to cytoplasmic ratios of Smad2/3 signal intensity is quantified in (e). n > 50 osteoblasts +/- transfer. (f) Quantification of the percent of SDF-1 expressing osteoblasts without co-culture, following 1 h of co-culture, and 5 h of co-culture. (n > 100 osteoblasts or +/- transfer scored for 3 independent experiments). (g) SDF-1 immunofluorescence of a transfer positive osteoblast. White lines outline the osteoblasts in the field of view. (h) Osteoblasts were co-cultured with N-Rh-PE labeled KG1a cells for 1 h and then washed to remove all KG1a cells. SDF-1 expression was detected by immunofluorescence immediately following KG1a cell removal and then 4 h following KG1a cell removal. (n > 100 transfer positive osteoblasts for 3 independent experiments). (i) To evaluate whether transfer rather than KG1a cell contact mediated the increase in SDF-1 expression, KG1a/osteoblast co-cultures were treated with 10 mM methyl β cyclodextrin (MβCD), 2 μm cytocholasin D (Cyto D), or 80 μm Dynasore to allow for KG1a cell contact with osteoblasts, but not transfer. Drug treatment, which reduced N-Rh-PE transfer (Fig. 3h), but allowed for cell contact, resulted in a decreased percent of SDF-1 expressing osteoblasts when compared to control co-cultures. (n > 600 osteoblasts for 3 independent experiments)

Mentions: To determine the significance of transferred molecule localization for cell communication between HPC and osteoblast, we further characterized the endocytic structures containing transferred molecules. In particular, we focused on the endocytic structures that were positive for the 2xFYVE domain-GFP, since many FYVE domain-containing proteins are involved in signal transduction [22]. SARA (Smad Anchor for Receptor Activation) is a FYVE domain-containing protein localized to endosomes, which correspond to a signaling compartment specialized for the propagation of extracellular signals such as TGFβ signaling [23, 24]. Upon TGFβ receptor activation, Smads are translocated to the nucleus with the help of the cofactor, SARA, resulting in gene activation. Osteoblasts fixed following N-Rh-PE transfer and stained using SARA-specific antibodies displayed a significant co-localization of N-Rh-PE with SARA-labeled endosomes (Fig. 5a). The histogram of the weighted co-localization coefficients (Fig. 5b) revealed a similar level of N-Rh-PE co-localization with SARA as with Rab 7, although SARA and Rab 7 appeared to represent two different populations of endosomes. These data showed that molecules transferred from the HPC membrane domain can be delivered to a SARA-positive signaling endosome within osteoblasts.


Intercellular transfer to signalling endosomes regulates an ex vivo bone marrow niche.

Gillette JM, Larochelle A, Dunbar CE, Lippincott-Schwartz J - Nat. Cell Biol. (2009)

Intercellular transfer correlates with decreased Smad 2/3 activation and an increased production of SDF-1 by the osteoblast. (a) N-Rh-PE (red) labeled KG1a cells were co-cultured for one hour with osteoblasts, which were then fixed and immunostained for SARA (green). Transferred N-Rh-PE could be detected within SARA positive vesicles in the osteoblast as indicated by the white circles. (b) Histogram of weighted co-localization coefficients for N-Rh-PE with a SARA positive endosome (n = 33 cells) and for SARA with Rab 7-GFP positive endosomes (n = 9 cells) indicates that transferred N-Rh-PE is detected within SARA positive endosomes, which are distinct from a Rab 7-GFP compartment. (c) Osteoblasts fixed and stained for Smad2/3 (green) have a high level of nuclear expression indicating activate Smad2/3. Following a transfer event, osteoblasts with detectable transfer (d) have a reduced nuclear localization of Smad2/3. The nuclear to cytoplasmic ratios of Smad2/3 signal intensity is quantified in (e). n > 50 osteoblasts +/- transfer. (f) Quantification of the percent of SDF-1 expressing osteoblasts without co-culture, following 1 h of co-culture, and 5 h of co-culture. (n > 100 osteoblasts or +/- transfer scored for 3 independent experiments). (g) SDF-1 immunofluorescence of a transfer positive osteoblast. White lines outline the osteoblasts in the field of view. (h) Osteoblasts were co-cultured with N-Rh-PE labeled KG1a cells for 1 h and then washed to remove all KG1a cells. SDF-1 expression was detected by immunofluorescence immediately following KG1a cell removal and then 4 h following KG1a cell removal. (n > 100 transfer positive osteoblasts for 3 independent experiments). (i) To evaluate whether transfer rather than KG1a cell contact mediated the increase in SDF-1 expression, KG1a/osteoblast co-cultures were treated with 10 mM methyl β cyclodextrin (MβCD), 2 μm cytocholasin D (Cyto D), or 80 μm Dynasore to allow for KG1a cell contact with osteoblasts, but not transfer. Drug treatment, which reduced N-Rh-PE transfer (Fig. 3h), but allowed for cell contact, resulted in a decreased percent of SDF-1 expressing osteoblasts when compared to control co-cultures. (n > 600 osteoblasts for 3 independent experiments)
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Figure 5: Intercellular transfer correlates with decreased Smad 2/3 activation and an increased production of SDF-1 by the osteoblast. (a) N-Rh-PE (red) labeled KG1a cells were co-cultured for one hour with osteoblasts, which were then fixed and immunostained for SARA (green). Transferred N-Rh-PE could be detected within SARA positive vesicles in the osteoblast as indicated by the white circles. (b) Histogram of weighted co-localization coefficients for N-Rh-PE with a SARA positive endosome (n = 33 cells) and for SARA with Rab 7-GFP positive endosomes (n = 9 cells) indicates that transferred N-Rh-PE is detected within SARA positive endosomes, which are distinct from a Rab 7-GFP compartment. (c) Osteoblasts fixed and stained for Smad2/3 (green) have a high level of nuclear expression indicating activate Smad2/3. Following a transfer event, osteoblasts with detectable transfer (d) have a reduced nuclear localization of Smad2/3. The nuclear to cytoplasmic ratios of Smad2/3 signal intensity is quantified in (e). n > 50 osteoblasts +/- transfer. (f) Quantification of the percent of SDF-1 expressing osteoblasts without co-culture, following 1 h of co-culture, and 5 h of co-culture. (n > 100 osteoblasts or +/- transfer scored for 3 independent experiments). (g) SDF-1 immunofluorescence of a transfer positive osteoblast. White lines outline the osteoblasts in the field of view. (h) Osteoblasts were co-cultured with N-Rh-PE labeled KG1a cells for 1 h and then washed to remove all KG1a cells. SDF-1 expression was detected by immunofluorescence immediately following KG1a cell removal and then 4 h following KG1a cell removal. (n > 100 transfer positive osteoblasts for 3 independent experiments). (i) To evaluate whether transfer rather than KG1a cell contact mediated the increase in SDF-1 expression, KG1a/osteoblast co-cultures were treated with 10 mM methyl β cyclodextrin (MβCD), 2 μm cytocholasin D (Cyto D), or 80 μm Dynasore to allow for KG1a cell contact with osteoblasts, but not transfer. Drug treatment, which reduced N-Rh-PE transfer (Fig. 3h), but allowed for cell contact, resulted in a decreased percent of SDF-1 expressing osteoblasts when compared to control co-cultures. (n > 600 osteoblasts for 3 independent experiments)
Mentions: To determine the significance of transferred molecule localization for cell communication between HPC and osteoblast, we further characterized the endocytic structures containing transferred molecules. In particular, we focused on the endocytic structures that were positive for the 2xFYVE domain-GFP, since many FYVE domain-containing proteins are involved in signal transduction [22]. SARA (Smad Anchor for Receptor Activation) is a FYVE domain-containing protein localized to endosomes, which correspond to a signaling compartment specialized for the propagation of extracellular signals such as TGFβ signaling [23, 24]. Upon TGFβ receptor activation, Smads are translocated to the nucleus with the help of the cofactor, SARA, resulting in gene activation. Osteoblasts fixed following N-Rh-PE transfer and stained using SARA-specific antibodies displayed a significant co-localization of N-Rh-PE with SARA-labeled endosomes (Fig. 5a). The histogram of the weighted co-localization coefficients (Fig. 5b) revealed a similar level of N-Rh-PE co-localization with SARA as with Rab 7, although SARA and Rab 7 appeared to represent two different populations of endosomes. These data showed that molecules transferred from the HPC membrane domain can be delivered to a SARA-positive signaling endosome within osteoblasts.

Bottom Line: Here, we use live-cell imaging to characterize both the site of contact between osteoblasts and haematopoietic progenitor cells (HPCs) and events at this site that result in downstream signalling responses important for niche maintenance.This caused osteoblasts to downregulate Smad signalling and increase production of stromal-derived factor-1 (SDF-1), a chemokine responsible for HSPC homing to bone marrow.These findings identify a mechanism involving intercellular transfer to signalling endosomes for targeted regulation of signalling and remodelling events within an ex vivo osteoblastic niche.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Program, National Institute of Child Health and Human Development, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892, USA.

ABSTRACT
Haematopoietic stem-progenitor cells (HSPCs) reside in the bone marrow niche, where interactions with osteoblasts provide essential cues for their proliferation and survival. Here, we use live-cell imaging to characterize both the site of contact between osteoblasts and haematopoietic progenitor cells (HPCs) and events at this site that result in downstream signalling responses important for niche maintenance. HPCs made prolonged contact with the osteoblast surface through a specialized membrane domain enriched in prominin 1, CD63 and rhodamine PE. At the contact site, portions of the specialized domain containing these molecules were taken up by the osteoblast and internalized into SARA-positive signalling endosomes. This caused osteoblasts to downregulate Smad signalling and increase production of stromal-derived factor-1 (SDF-1), a chemokine responsible for HSPC homing to bone marrow. These findings identify a mechanism involving intercellular transfer to signalling endosomes for targeted regulation of signalling and remodelling events within an ex vivo osteoblastic niche.

Show MeSH
Related in: MedlinePlus