Nucleolar localization and identification of nuclear/nucleolar localization signals of the calmodulin-binding protein nucleomorphin during growth and mitosis in Dictyostelium

Catalano, Andrew; O’Day, Danton H.

doi:10.1007/s00418-011-0785-3

Cited by 17 publications

(31 citation statements)

References 65 publications

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“…-binding domain, NES, and NLS from these different organisms. c Diagram of all GFP-fusion proteins used in this study (Catalano and O'Day 2011b). In order to determine if the localization of PsaA is dependent on either proper NumA1 localization or on transcription, cells were treated with AM-D prior to immunolocalization with anti-PsaA.…”

Section: No Effect Of Transcription Inhibition On Psaa Localizationmentioning

confidence: 99%

“…For detection of GFP-fusion protein by anti-Psa, cells expressing either GFP, N-, or C-GFP-PsaA were probed with anti-PsaA followed by Alexa Fluor Ò 555 goat anti-rabbit. For colocalization of PsaA and NumA1, C-GFP-PsaA-expressing cells were probed with anti-NumA1 (1:40) (Catalano and O'Day 2011b;O'Day et al 2009) followed by Alexa Fluor Ò 555 goat anti-rabbit (1:40). The localization of a-tubulin was used to determine the stage of cell cycle and cell integrity.…”

Section: Antibody Productionmentioning

confidence: 99%

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Dictyostelium puromycin-sensitive aminopeptidase A is a nucleoplasmic nucleomorphin-binding protein that relocates to the cytoplasm during mitosis

Catalano

Poloz

O’Day

2011

Histochem Cell Biol

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Nucleomorphin (NumA1) is a nucleolar/nucleoplasmic protein linked to cell cycle in Dictyostelium. It interacts with puromycin-sensitive aminopeptidase A (PsaA) which in other organisms is a Zn(2+)-metallopeptidase thought to be involved in cell cycle progression and is involved in several human diseases. Here, we have shown that Dictyostelium PsaA contains domains characteristic of the M1 family of Zn(2+)-metallopeptidases: a GAMEN motif and a Zn(2+)-binding domain. PsaA colocalized with NumA1 in the nucleoplasm in vegetative cells and was also present to a lesser extent in the cytoplasm. The same localization pattern was observed in cells from slugs, however, in fruiting bodies PsaA was only detected in spore nuclei. During mitosis PsaA redistributed mainly throughout the cytoplasm. It possesses a functional nuclear localization signal ((680)RKRF(683)) necessary for nuclear entry. To our knowledge, this is the first nuclear localization signal identified in a Psa from any organism. Treatment with Ca(2+) chelators or calmodulin antagonists indicated that neither Ca(2+) nor calmodulin is involved in PsaA localization. These results are interpreted in terms of the inter-relationship between NumA1 and PsaA in cell function in Dictyostelium.

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Section: No Effect Of Transcription Inhibition On Psaa Localizationmentioning

confidence: 99%

Section: Antibody Productionmentioning

confidence: 99%

Dictyostelium puromycin-sensitive aminopeptidase A is a nucleoplasmic nucleomorphin-binding protein that relocates to the cytoplasm during mitosis

Catalano

Poloz

O’Day

2011

Histochem Cell Biol

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“…NumA1 is an acidic, nucleolar Ca 2ϩ -dependent calmodulin-binding protein (CaMBP) (33). Several pieces of evidence suggest that it regulates cell cycle in D. discoideum (6,32,33). NumA1 is also expressed throughout development, its expression is regulated by DIF-1, and it interacts with a prestalk O (pstO)-specific protein Ca 2ϩ -binding protein 4a (CBP4a) (32,34,37).…”

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confidence: 99%

Bestatin Inhibits Cell Growth, Cell Division, and Spore Cell Differentiation in Dictyostelium discoideum

2012

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ABSTRACTBestatin methyl ester (BME) is an inhibitor of Zn2+-binding aminopeptidases that inhibits cell proliferation and induces apoptosis in normal and cancer cells. We have usedDictyosteliumas a model organism to study the effects of BME. Only two Zn2+-binding aminopeptidases have been identified inDictyosteliumto date, puromycin-sensitive aminopeptidase A and B (PsaA and PsaB). PSA from other organisms is known to regulate cell division and differentiation. Here we show that PsaA is differentially expressed throughout growth and development ofDictyostelium, and its expression is regulated by developmental morphogens. We present evidence that BME specifically interacts with PsaA and inhibits its aminopeptidase activity. Treatment of cells with BME inhibited the rate of cell growth and the frequency of cell division in growing cells and inhibited spore cell differentiation during late development. Overexpression of PsaA-GFP (where GFP is green fluorescent protein) also inhibited spore cell differentiation but did not affect growth. Using chimeras, we have identified that nuclear versus cytoplasmic localization of PsaA affects the choice between stalk or spore cell differentiation pathway. Cells that overexpressed PsaA-GFP (primarily nuclear) differentiated into stalk cells, while cells that overexpressed PsaAΔNLS2-GFP (cytoplasmic) differentiated into spores. In conclusion, we have identified that BME inhibits cell growth, division, and differentiation inDictyosteliumlikely through inhibition of PsaA.

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“…Anti-NumA1, anti-CBP4a, and anti-Snf12 were produced and verified as previously described [15][16][17]19]. Cell lines expressing either C-GFP-Snf12 or GFP-NLS-3 (GFP-KRKR) from Snf12 were made and verified as previously described [17].…”

Section: Antibodies and Gfp-fusion Protein-expressing Cell Linesmentioning

confidence: 99%

“…Calcium-binding protein 4a (CBP4a) and forkhead-associated kinase A (fhkA) are two recently identified proteins in Dictyostelium that localize to different nucleolar locales; FhkA localizes around the nucleolar periphery while CBP4a resides within the nucleoplasm [15]. BAF60a/SMARCD1 homologue Snf12 and NumA1, two nucleolar proteins linked to cell cycle in Dictyostelium, localize differently since Snf12 localizes to a smaller area of the nucleolus than NumA1 [16,17]. If, as this work suggests, functional subcompartments do exist, these and other nucleolar proteins should localize to different regions of the nucleolus, thereby delineating some of the nucleolar subcompartment locales.…”

Section: Introductionmentioning

confidence: 99%

Evidence for nucleolar subcompartments in Dictyostelium

Catalano

O’Day

2015

Biochemical and Biophysical Research Communications

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The nucleolus is a multifunctional nuclear compartment usually consisting of two to three subcompartments which represent stages of ribosomal biogenesis. It is linked to several human diseases including viral infections, cancer, and neurodegeneration. Dictyostelium is a model eukaryote for the study of fundamental biological processes as well as several human diseases however comparatively little is known about its nucleolus. Unlike most nucleoli it does not possess visible subcompartments at the ultrastructural level. Several recently identified nucleolar proteins in Dictyostelium leave the nucleolus after treatment with the rDNA transcription inhibitor actinomycin-D (AM-D). Different proteins exit in different ways, suggesting that previously unidentified nucleolar subcompartments may exist. The identification of nucleolar subcompartments would help to better understand the nucleolus in this model eukaryote. Here, we show that Dictyostelium nucleolar proteins nucleomorphin isoform NumA1 and Bud31 localize throughout the entire nucleolus while calcium-binding protein 4a localizes to only a portion, representing nucleolar subcompartment 1 (NoSC1). SWI/SNF complex member Snf12 localizes to a smaller area within NoSC1 representing a second nucleolar subcompartment, NoSC2. The nuclear/nucleolar localization signal KRKR from Snf12 localized GFP to NoSC2, and thus also appears to function as a nucleolar subcompartment localization signal. FhkA localizes to the nucleolar periphery displaying a similar pattern to that of Hsp32. Similarities between the redistribution patterns of Dictyostelium nucleolar proteins during nucleolar disruption as a result of either AM-D treatment or mitosis support these subcompartments. A model for the AM-D-induced redistribution patterns is proposed.

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Nucleolar localization and identification of nuclear/nucleolar localization signals of the calmodulin-binding protein nucleomorphin during growth and mitosis in Dictyostelium

Cited by 17 publications

References 65 publications

Dictyostelium puromycin-sensitive aminopeptidase A is a nucleoplasmic nucleomorphin-binding protein that relocates to the cytoplasm during mitosis

Dictyostelium puromycin-sensitive aminopeptidase A is a nucleoplasmic nucleomorphin-binding protein that relocates to the cytoplasm during mitosis

Bestatin Inhibits Cell Growth, Cell Division, and Spore Cell Differentiation in Dictyostelium discoideum

Evidence for nucleolar subcompartments in Dictyostelium

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