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First published online January 12, 2006
doi: 10.1242/10.1242/jcs.02753

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The Shwachman-Bodian-Diamond syndrome gene encodes an RNA-binding protein that localizes to the pseudopod of Dictyostelium amoebae during chemotaxis

¹ W.M. Keck Dynamic Image Analysis Facility, Department of Biological Sciences, The University of Iowa, Iowa City, IA 52242, USA
² Computational Biology Branch, NCBI, NLM, NIH, Bethesda, MD 20894, USA

View larger version (33K): [in a new window]   Fig. 1. Alignment of human and D. discoideum SBDS proteins. The boundaries of the three major domains in SBDS and related orthologs include: an N-terminal module with two motifs, SHHP1 and SHHP2; a central tri-helical bundle module; and a C-terminal module with RNA-recognition motif (RRM-like). The four ß strands (E-runs) and four helices (H-runs) in the N-terminal module, and the unique 24-residue tail of the Dictyostelium SBDS ortholog are indicated.

View larger version (64K): [in a new window]   Fig. 2. Northern analysis of SBDS-GFP using the GFP and the SBDS sequences as probes.

View larger version (33K): [in a new window]   Fig. 3. The SBDS protein is distributed relatively evenly throughout the cytoplasm and pseudopod of D. discoideum amoebae that migrate in buffer, but is enriched in the anterior pseudopods of cells that undergo chemotaxis in a spatial gradient of chemoattractant. Cells carrying GFP-tagged SBDS were imaged with LSCM 1 µm above the substratum. (A,B,C) Examples of the even distribution of SBDS-GFP in cells that migrate in buffer. (D) Images of a single cell migrating in buffer, taken every 10 seconds and 1 µm above the substratum. (E,F,G) Examples of the enrichment of SBDS-GFP in the pseudopods of cells undergoing chemotaxis. (H) Images of a single cell undergoing chemotaxis, taken every 10 seconds and 1 µm above the substratum. Arrows represent the direction of increasing chemoattractant concentration; a, anterior end; u, uropod. Bars, 5 µm (bar in A also represents B, D; bar in E also represents F, H).

View larger version (37K): [in a new window]   Fig. 4. Pixel intensity of line scans made with the LSCM across different parts of the cell. Each data point is the average of the line scan taken as the cell either migrates in buffer or undergoes chemotaxis. (A) Diagram of line scans and color code of different parts of cell body. (B-E) Examples of cells migrating in buffer. (F-I) Examples of cells undergoing chemotaxis in a spatial gradient of attractant. Notice that the average pixel intensity across pseudopods is similar to that of the cell body in buffer, but consistently higher in cells undergoing chemotaxis.

View larger version (41K): [in a new window]   Fig. 5. SBDS-GFP localizes to the site of incipient lateral pseudopod formation, remains localized in the pseudopod during extension and exits the pseudopod upon retraction. (A,B) Two representative examples of localization. Small arrows point to SBDS-GFP localization in lateral pseudopods. Large arrows indicate direction of spatial gradients. Time is given in seconds (s); a, anterior end; u, uropod. Bar, 5 µm.

View larger version (13K): [in a new window]   Fig. 6. SBDS-GFP localizes to the pseudopods of cells treated in a perfusion chamber with a constant concentration of chemoattractant (cAMP). (A,B) Cells were perfused before imaging with (A) buffer in the absence of cAMP for 10 minutes or (B,C) buffer containing 5x10–8 M cAMP for 10 minutes; a, anterior end; u, uropod. Bar, 5 µm.

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