Limits...
Altered intracellular localization and mobility of SBDS protein upon mutation in Shwachman-Diamond syndrome.

Orelio C, van der Sluis RM, Verkuijlen P, Nethe M, Hordijk PL, van den Berg TK, Kuijpers TW - PLoS ONE (2011)

Bottom Line: Further studies with a series of SBDS mutant proteins revealed that three distinct motifs determine the intracellular mobility of SBDS protein.A sumoylation motif in the C-terminal domain, that is lacking in patient SBDS proteins, was found to play a pivotal role in intracellular motility.Our structure-function analyses provide new insight into localization and motility of the SBDS protein, and show that patient-related mutant proteins are altered in their molecular properties, which may contribute to the clinical features observed in SDS patients.

View Article: PubMed Central - PubMed

Affiliation: Sanquin Research and Landsteiner Laboratory of the Academic Medical Center, Department of Blood Cell Research, University of Amsterdam, Amsterdam, The Netherlands.

ABSTRACT
Shwachman-Diamond Syndrome (SDS) is a rare inherited disease caused by mutations in the SBDS gene. Hematopoietic defects, exocrine pancreas dysfunction and short stature are the most prominent clinical features. To gain understanding of the molecular properties of the ubiquitously expressed SBDS protein, we examined its intracellular localization and mobility by live cell imaging techniques. We observed that SBDS full-length protein was localized in both the nucleus and cytoplasm, whereas patient-related truncated SBDS protein isoforms localize predominantly to the nucleus. Also the nucleo-cytoplasmic trafficking of these patient-related SBDS proteins was disturbed. Further studies with a series of SBDS mutant proteins revealed that three distinct motifs determine the intracellular mobility of SBDS protein. A sumoylation motif in the C-terminal domain, that is lacking in patient SBDS proteins, was found to play a pivotal role in intracellular motility. Our structure-function analyses provide new insight into localization and motility of the SBDS protein, and show that patient-related mutant proteins are altered in their molecular properties, which may contribute to the clinical features observed in SDS patients.

Show MeSH

Related in: MedlinePlus

SDS-patient SBDS proteins have different intracellular mobility characteristics compared to GFP-SBDS-FL.(A) Representative GFP-SBDS-FL and (B) GFP-SBDS-C84-expressing cell for FRAP analysis prior to bleaching, at the moment of nuclear bleaching and 10 min post-bleach. (C) FRAP analysis showing the average nuclear/cytoplasmic GFP ratio for free GFP (black curve), GFP-SBDS-FL, GFP-SBDS-C84 and GFP-SBDS-R218 (grey curves); 5–9 cells per construct in 2–3 independent experiments were analysed for 10 min recovery and 12–14 cells for 5 min recovery in 3 independent experiments (not shown). (D) Representative GFP-SBDS-FL and (E) Representative GFP-SBDS-C84 expressing cell for FLIP analysis prior to bleaching, at the moment of nuclear bleaching and 10 min post-bleach (F) FLIP analysis showing the average nuclear GFP intensity for free GFP (black curve), GFP-SBDS-FL, GFP-SBDS-C84 and GFP-SBDS-R218; 7–11 cells per construct in 2–3 independent experiments were analysed.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3113850&req=5

pone-0020727-g002: SDS-patient SBDS proteins have different intracellular mobility characteristics compared to GFP-SBDS-FL.(A) Representative GFP-SBDS-FL and (B) GFP-SBDS-C84-expressing cell for FRAP analysis prior to bleaching, at the moment of nuclear bleaching and 10 min post-bleach. (C) FRAP analysis showing the average nuclear/cytoplasmic GFP ratio for free GFP (black curve), GFP-SBDS-FL, GFP-SBDS-C84 and GFP-SBDS-R218 (grey curves); 5–9 cells per construct in 2–3 independent experiments were analysed for 10 min recovery and 12–14 cells for 5 min recovery in 3 independent experiments (not shown). (D) Representative GFP-SBDS-FL and (E) Representative GFP-SBDS-C84 expressing cell for FLIP analysis prior to bleaching, at the moment of nuclear bleaching and 10 min post-bleach (F) FLIP analysis showing the average nuclear GFP intensity for free GFP (black curve), GFP-SBDS-FL, GFP-SBDS-C84 and GFP-SBDS-R218; 7–11 cells per construct in 2–3 independent experiments were analysed.

Mentions: First, FRAP analysis of GFP-SBDS-FL within the nucleus and the cytoplasm revealed that GFP-SBDS-FL protein levels were minimally recovered to the nucleus within 10 minutes post-bleaching. GFP-SBDS-FL distribution remained similar to the moment immediately after bleaching (3% of original intensity; Fig. 2A/C and Suppl. Fig. S2A). In contrast to GFP-SBDS-FL, we observed that free GFP was rapidly redistributed from the cytoplasm to the nucleus after nuclear bleaching. Within 5–6 min GFP was distributed intracellular in a similar fashion as pre-bleaching (Fig. 2C), which is consistent with the rate of free GFP mobility previously reported by others [29]. To further investigate the striking lack of nuclear import of the GFP-SBDS-FL protein, we examined the possibility that GFP-SBDS-FL nuclear import and export was hampered due to immobility in either the cytoplasm or the nucleus as the results of protein-protein interactions. Therefore, we bleached part of the cytoplasm and monitored fluorescence recovery. Within 2 minutes post-bleach full cytoplasmic recovery at the bleach area was observed, showing that GFP-SBDS-FL is mobile within the cytoplasm (Suppl. Fig. S2B), although the recovery kinetics of GFP-SBDS-FL is slower than GFP (data not shown). Similar experiments were performed to examine SBDS nuclear mobility. We observed that also within the nucleus GFP-SBDS-FL was mobile (data not shown). To examine whether eventually GFP-SBDS-FL fluorescence could be recovered after nuclear bleaching, we performed imaging for several hours after bleaching. We observed that GFP-SBDS-FL fluorescence in the nucleus recovers only after 1–2 hours post nuclear bleaching, indicating that GFP-SBDS-FL nucleo-cytoplasmic transport under steady state conditions takes place at an extremely slow rate (n = 17 cells; 2 independent experiments; data not shown).


Altered intracellular localization and mobility of SBDS protein upon mutation in Shwachman-Diamond syndrome.

Orelio C, van der Sluis RM, Verkuijlen P, Nethe M, Hordijk PL, van den Berg TK, Kuijpers TW - PLoS ONE (2011)

SDS-patient SBDS proteins have different intracellular mobility characteristics compared to GFP-SBDS-FL.(A) Representative GFP-SBDS-FL and (B) GFP-SBDS-C84-expressing cell for FRAP analysis prior to bleaching, at the moment of nuclear bleaching and 10 min post-bleach. (C) FRAP analysis showing the average nuclear/cytoplasmic GFP ratio for free GFP (black curve), GFP-SBDS-FL, GFP-SBDS-C84 and GFP-SBDS-R218 (grey curves); 5–9 cells per construct in 2–3 independent experiments were analysed for 10 min recovery and 12–14 cells for 5 min recovery in 3 independent experiments (not shown). (D) Representative GFP-SBDS-FL and (E) Representative GFP-SBDS-C84 expressing cell for FLIP analysis prior to bleaching, at the moment of nuclear bleaching and 10 min post-bleach (F) FLIP analysis showing the average nuclear GFP intensity for free GFP (black curve), GFP-SBDS-FL, GFP-SBDS-C84 and GFP-SBDS-R218; 7–11 cells per construct in 2–3 independent experiments were analysed.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113850&req=5

pone-0020727-g002: SDS-patient SBDS proteins have different intracellular mobility characteristics compared to GFP-SBDS-FL.(A) Representative GFP-SBDS-FL and (B) GFP-SBDS-C84-expressing cell for FRAP analysis prior to bleaching, at the moment of nuclear bleaching and 10 min post-bleach. (C) FRAP analysis showing the average nuclear/cytoplasmic GFP ratio for free GFP (black curve), GFP-SBDS-FL, GFP-SBDS-C84 and GFP-SBDS-R218 (grey curves); 5–9 cells per construct in 2–3 independent experiments were analysed for 10 min recovery and 12–14 cells for 5 min recovery in 3 independent experiments (not shown). (D) Representative GFP-SBDS-FL and (E) Representative GFP-SBDS-C84 expressing cell for FLIP analysis prior to bleaching, at the moment of nuclear bleaching and 10 min post-bleach (F) FLIP analysis showing the average nuclear GFP intensity for free GFP (black curve), GFP-SBDS-FL, GFP-SBDS-C84 and GFP-SBDS-R218; 7–11 cells per construct in 2–3 independent experiments were analysed.
Mentions: First, FRAP analysis of GFP-SBDS-FL within the nucleus and the cytoplasm revealed that GFP-SBDS-FL protein levels were minimally recovered to the nucleus within 10 minutes post-bleaching. GFP-SBDS-FL distribution remained similar to the moment immediately after bleaching (3% of original intensity; Fig. 2A/C and Suppl. Fig. S2A). In contrast to GFP-SBDS-FL, we observed that free GFP was rapidly redistributed from the cytoplasm to the nucleus after nuclear bleaching. Within 5–6 min GFP was distributed intracellular in a similar fashion as pre-bleaching (Fig. 2C), which is consistent with the rate of free GFP mobility previously reported by others [29]. To further investigate the striking lack of nuclear import of the GFP-SBDS-FL protein, we examined the possibility that GFP-SBDS-FL nuclear import and export was hampered due to immobility in either the cytoplasm or the nucleus as the results of protein-protein interactions. Therefore, we bleached part of the cytoplasm and monitored fluorescence recovery. Within 2 minutes post-bleach full cytoplasmic recovery at the bleach area was observed, showing that GFP-SBDS-FL is mobile within the cytoplasm (Suppl. Fig. S2B), although the recovery kinetics of GFP-SBDS-FL is slower than GFP (data not shown). Similar experiments were performed to examine SBDS nuclear mobility. We observed that also within the nucleus GFP-SBDS-FL was mobile (data not shown). To examine whether eventually GFP-SBDS-FL fluorescence could be recovered after nuclear bleaching, we performed imaging for several hours after bleaching. We observed that GFP-SBDS-FL fluorescence in the nucleus recovers only after 1–2 hours post nuclear bleaching, indicating that GFP-SBDS-FL nucleo-cytoplasmic transport under steady state conditions takes place at an extremely slow rate (n = 17 cells; 2 independent experiments; data not shown).

Bottom Line: Further studies with a series of SBDS mutant proteins revealed that three distinct motifs determine the intracellular mobility of SBDS protein.A sumoylation motif in the C-terminal domain, that is lacking in patient SBDS proteins, was found to play a pivotal role in intracellular motility.Our structure-function analyses provide new insight into localization and motility of the SBDS protein, and show that patient-related mutant proteins are altered in their molecular properties, which may contribute to the clinical features observed in SDS patients.

View Article: PubMed Central - PubMed

Affiliation: Sanquin Research and Landsteiner Laboratory of the Academic Medical Center, Department of Blood Cell Research, University of Amsterdam, Amsterdam, The Netherlands.

ABSTRACT
Shwachman-Diamond Syndrome (SDS) is a rare inherited disease caused by mutations in the SBDS gene. Hematopoietic defects, exocrine pancreas dysfunction and short stature are the most prominent clinical features. To gain understanding of the molecular properties of the ubiquitously expressed SBDS protein, we examined its intracellular localization and mobility by live cell imaging techniques. We observed that SBDS full-length protein was localized in both the nucleus and cytoplasm, whereas patient-related truncated SBDS protein isoforms localize predominantly to the nucleus. Also the nucleo-cytoplasmic trafficking of these patient-related SBDS proteins was disturbed. Further studies with a series of SBDS mutant proteins revealed that three distinct motifs determine the intracellular mobility of SBDS protein. A sumoylation motif in the C-terminal domain, that is lacking in patient SBDS proteins, was found to play a pivotal role in intracellular motility. Our structure-function analyses provide new insight into localization and motility of the SBDS protein, and show that patient-related mutant proteins are altered in their molecular properties, which may contribute to the clinical features observed in SDS patients.

Show MeSH
Related in: MedlinePlus