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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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Transferred molecules are detected within various endocytic compartments of the osteoblasts. Live cell microscopy of N-Rh-PE (red) labeled KG1a cells co-cultured with osteoblastic cells transiently transfected with (a) Rab 5-GFP (green) or (b) Rab 7-GFP (green). White circles indicate transferred N-Rh-PE localized to a Rab 5-GFP or a Rab 7-GFP positive vesicle. Arrows indicate transferred N-Rh-PE not present in a Rab 5- or Rab 7-GFP vesicle. The osteoblast edge is outlined in white. (c) Histogram of weighted co-localization coefficients. As a positive control for a weighted co-localization coefficient, osteoblasts were transfected with EEA1-YFP and immunostained with an EEA-1 antibody (positive control, n = 6 cells). For a negative control, osteoblasts were transiently transfected with clathrin light chain-YFP and co-cultured with KG1a cells labeled with N-Rh-PE to allow for transfer. Weighted co-localization coefficients were calculated three hours following transfer of N-Rh-PE (negative control, n = 6 cells). All other co-localization coefficients were calculated following 1 h of co-culture (n = 20 cells for each condition). (d) Live cell microscopy of N-Rh-PE (red) labeled KG1a cells co-cultured with osteoblastic cells transiently transfected with 2xFYVE-GFP (green). White circles indicate transferred N-Rh-PE localized to a 2xFYVE-GFP positive vesicle. Both the KG1a cell and the contacted osteoblast are outlined in white. (e) Live-cell confocal imaging of osteoblasts following prominin 1-GFP (red) transfer from a transiently transfected KG1a cell. Before imaging, osteoblasts were washed with fresh medium to remove all KG1a cells, so only transferred prominin 1-GFP was detected. (f) Quantification of the relative fluorescence intensities over the course of 12 h for transferred prominin 1-GFP (solid line, n = 3 cells), internalized soluble amyloid beta-488 (dashed line, n = 6 cells), and internalized rhodamine-EGF (dotted line, n = 6 cells). Scale bars – 5μm.
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Figure 4: Transferred molecules are detected within various endocytic compartments of the osteoblasts. Live cell microscopy of N-Rh-PE (red) labeled KG1a cells co-cultured with osteoblastic cells transiently transfected with (a) Rab 5-GFP (green) or (b) Rab 7-GFP (green). White circles indicate transferred N-Rh-PE localized to a Rab 5-GFP or a Rab 7-GFP positive vesicle. Arrows indicate transferred N-Rh-PE not present in a Rab 5- or Rab 7-GFP vesicle. The osteoblast edge is outlined in white. (c) Histogram of weighted co-localization coefficients. As a positive control for a weighted co-localization coefficient, osteoblasts were transfected with EEA1-YFP and immunostained with an EEA-1 antibody (positive control, n = 6 cells). For a negative control, osteoblasts were transiently transfected with clathrin light chain-YFP and co-cultured with KG1a cells labeled with N-Rh-PE to allow for transfer. Weighted co-localization coefficients were calculated three hours following transfer of N-Rh-PE (negative control, n = 6 cells). All other co-localization coefficients were calculated following 1 h of co-culture (n = 20 cells for each condition). (d) Live cell microscopy of N-Rh-PE (red) labeled KG1a cells co-cultured with osteoblastic cells transiently transfected with 2xFYVE-GFP (green). White circles indicate transferred N-Rh-PE localized to a 2xFYVE-GFP positive vesicle. Both the KG1a cell and the contacted osteoblast are outlined in white. (e) Live-cell confocal imaging of osteoblasts following prominin 1-GFP (red) transfer from a transiently transfected KG1a cell. Before imaging, osteoblasts were washed with fresh medium to remove all KG1a cells, so only transferred prominin 1-GFP was detected. (f) Quantification of the relative fluorescence intensities over the course of 12 h for transferred prominin 1-GFP (solid line, n = 3 cells), internalized soluble amyloid beta-488 (dashed line, n = 6 cells), and internalized rhodamine-EGF (dotted line, n = 6 cells). Scale bars – 5μm.

Mentions: We next followed the fate of transferred material taken up by the osteoblast. After 30 min of uptake, HPC components were seen in various osteoblast endocytic compartments, including Rab5- and Rab7-positive structures (Fig, 4a-c). In addition, HPC components were observed in endocytic structures positive for the 2xFYVE domain [20] (Fig. 4c,d). Imaging of single osteoblasts that had taken up prominin 1-GFP from a contacting HPC (Fig. 4e) showed that the transferred molecules remained in distinct intracellular structures within the osteoblast for up to 12 h post-transfer. In contrast, endocytosed EGF and the soluble amyloid beta protein, which are delivered to lysosomes for degradation [21], were completely destroyed by the osteoblast during a similar time period (Fig. 4f). These results suggested that while transferred molecules could be degraded, a proportion of these molecules avoided destruction within osteoblast lysosomes and were delivered through endocytic intermediates to a longer-lived intracellular compartment.


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)

Transferred molecules are detected within various endocytic compartments of the osteoblasts. Live cell microscopy of N-Rh-PE (red) labeled KG1a cells co-cultured with osteoblastic cells transiently transfected with (a) Rab 5-GFP (green) or (b) Rab 7-GFP (green). White circles indicate transferred N-Rh-PE localized to a Rab 5-GFP or a Rab 7-GFP positive vesicle. Arrows indicate transferred N-Rh-PE not present in a Rab 5- or Rab 7-GFP vesicle. The osteoblast edge is outlined in white. (c) Histogram of weighted co-localization coefficients. As a positive control for a weighted co-localization coefficient, osteoblasts were transfected with EEA1-YFP and immunostained with an EEA-1 antibody (positive control, n = 6 cells). For a negative control, osteoblasts were transiently transfected with clathrin light chain-YFP and co-cultured with KG1a cells labeled with N-Rh-PE to allow for transfer. Weighted co-localization coefficients were calculated three hours following transfer of N-Rh-PE (negative control, n = 6 cells). All other co-localization coefficients were calculated following 1 h of co-culture (n = 20 cells for each condition). (d) Live cell microscopy of N-Rh-PE (red) labeled KG1a cells co-cultured with osteoblastic cells transiently transfected with 2xFYVE-GFP (green). White circles indicate transferred N-Rh-PE localized to a 2xFYVE-GFP positive vesicle. Both the KG1a cell and the contacted osteoblast are outlined in white. (e) Live-cell confocal imaging of osteoblasts following prominin 1-GFP (red) transfer from a transiently transfected KG1a cell. Before imaging, osteoblasts were washed with fresh medium to remove all KG1a cells, so only transferred prominin 1-GFP was detected. (f) Quantification of the relative fluorescence intensities over the course of 12 h for transferred prominin 1-GFP (solid line, n = 3 cells), internalized soluble amyloid beta-488 (dashed line, n = 6 cells), and internalized rhodamine-EGF (dotted line, n = 6 cells). Scale bars – 5μm.
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Figure 4: Transferred molecules are detected within various endocytic compartments of the osteoblasts. Live cell microscopy of N-Rh-PE (red) labeled KG1a cells co-cultured with osteoblastic cells transiently transfected with (a) Rab 5-GFP (green) or (b) Rab 7-GFP (green). White circles indicate transferred N-Rh-PE localized to a Rab 5-GFP or a Rab 7-GFP positive vesicle. Arrows indicate transferred N-Rh-PE not present in a Rab 5- or Rab 7-GFP vesicle. The osteoblast edge is outlined in white. (c) Histogram of weighted co-localization coefficients. As a positive control for a weighted co-localization coefficient, osteoblasts were transfected with EEA1-YFP and immunostained with an EEA-1 antibody (positive control, n = 6 cells). For a negative control, osteoblasts were transiently transfected with clathrin light chain-YFP and co-cultured with KG1a cells labeled with N-Rh-PE to allow for transfer. Weighted co-localization coefficients were calculated three hours following transfer of N-Rh-PE (negative control, n = 6 cells). All other co-localization coefficients were calculated following 1 h of co-culture (n = 20 cells for each condition). (d) Live cell microscopy of N-Rh-PE (red) labeled KG1a cells co-cultured with osteoblastic cells transiently transfected with 2xFYVE-GFP (green). White circles indicate transferred N-Rh-PE localized to a 2xFYVE-GFP positive vesicle. Both the KG1a cell and the contacted osteoblast are outlined in white. (e) Live-cell confocal imaging of osteoblasts following prominin 1-GFP (red) transfer from a transiently transfected KG1a cell. Before imaging, osteoblasts were washed with fresh medium to remove all KG1a cells, so only transferred prominin 1-GFP was detected. (f) Quantification of the relative fluorescence intensities over the course of 12 h for transferred prominin 1-GFP (solid line, n = 3 cells), internalized soluble amyloid beta-488 (dashed line, n = 6 cells), and internalized rhodamine-EGF (dotted line, n = 6 cells). Scale bars – 5μm.
Mentions: We next followed the fate of transferred material taken up by the osteoblast. After 30 min of uptake, HPC components were seen in various osteoblast endocytic compartments, including Rab5- and Rab7-positive structures (Fig, 4a-c). In addition, HPC components were observed in endocytic structures positive for the 2xFYVE domain [20] (Fig. 4c,d). Imaging of single osteoblasts that had taken up prominin 1-GFP from a contacting HPC (Fig. 4e) showed that the transferred molecules remained in distinct intracellular structures within the osteoblast for up to 12 h post-transfer. In contrast, endocytosed EGF and the soluble amyloid beta protein, which are delivered to lysosomes for degradation [21], were completely destroyed by the osteoblast during a similar time period (Fig. 4f). These results suggested that while transferred molecules could be degraded, a proportion of these molecules avoided destruction within osteoblast lysosomes and were delivered through endocytic intermediates to a longer-lived intracellular compartment.

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