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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 1, 2004 as doi:10.1096/fj.03-1459fje. |
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* Departmento de Biologia, Instituto de Biociências, Universidade de São Paulo, São Paulo;
Laboratório de Toxinologia Molecular, Instituto Butantan, São Paulo; and
Departmento de Bioquímica e Imunologia, Universidade de São Paulo, Ribeirão Preto, SP, Brasil
1Correspondence: Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, 277, 05508-900, São Paulo, SP, Brasil. E-mail: akerkis{at}usp.br
SPECIFIC AIMS
Peptides rich in basic arginine and lysine residues can be internalized by cells in vitro and in vivo and have been used for intracellular delivery of genes, therapeutic agents, and diagnostic probes. Crotamine, a toxin from the venom of the South American rattlesnake, is a 42 amino acid cationic polypeptide (YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWKCCKKGS—G)containing 11 basic residues (9 lysines, 2 arginines) and 6 cysteines. We evaluated the cytotoxicity of crotamine in murine ES cells. Embryotoxicity was tested in vitro in preimplantation stages (morulae/blastocysts) of the mouse embryo. Uptake of Cy3-conjugated crotamine (Cy3-crotamine) and its intracellular localization were analyzed in different human cells, murine undifferentiated and differentiating ES cells, and in vivo in mouse cells from bone marrow, spleen, and the peritoneal cavity.
PRINCIPAL FINDINGS
1. Nontoxic concentrations of crotamine
Since pluripotency is a conspicuous feature of ES cells that can be lost in altered culture conditions, the effect of 10 and 0.1 µM crotamine on the pluripotency of mouse ES cell line USP-1 was tested during three passages. Alkaline phosphatase was active in undifferentiated cells and its activity was not altered when ES cells were maintained under crotamine treatment. The dynamics of embryoid body (Eb) formation were not impaired, as shown by the number of developed Ebs in experimental and control conditions, as well as by typical rhythmic contractions of the heart muscle observed on developmental day 8. The susceptibility of murine ES and embryonic mouse fibroblasts to crotamine concentrations of 1000–0.01 µM was assayed by clonogenicity. At concentrations of 
10 µM, crotamine exhibited no toxic effects even after 72 h of exposure, and crotamine-treated cells proliferated for >10 passages. Embryotoxicity of crotamine was investigated by incubating 2.5 day postcoitum compact mouse morulae in the presence of 1 µM crotamine for 24 h. The number of compact morulae developing into blastocysts was similar in control and test groups.
2. In vitro and in vivo uptake of Cy3-crotamine
Cellular uptake of Cy3-crotamine at the nontoxic concentration of 1 µM was monitored by confocal microscopy after 5 min and 1, 3, 24, and 48 h treatment.
In vitro crotamine uptake was observed in human primary fibroblasts, lymphoblastic cells, murine embryonic stem (pluripotent and differentiated), and endothelial s-vec cells. Human fibroblasts were intensively labeled after 1 h of incubation with crotamine (Fig. 1
A); supposedly dividing cells showed stronger signals (Fig. 1B, C
). Pluripotent murine ES cells showed strong fluorescence within islands composed of rounded-up, juxtaposed cells growing onto a feeder layer of inactivated mouse fibroblasts; some islands were weakly labeled (Fig. 1D, E
). A weak background of fluorescent signals was seen in control ES cultures stained by Cy3 dye (Fig. 1F
).
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In vivo uptake of crotamine was studied by i.p. injection of Cy3-crotamine into CD-1 mice. Strong fluorescence was observed in peritoneal liquid cells (Fig. 1G-I
) and bone marrow cells; only weak background fluorescence was seen in control Cy3-injected mice.
The cells internalize crotamine within 5 min after its addition. The number of labeled cells did not appear to increase with longer exposure times, reaching a maximum after 
3 h of treatment. Cell labeling was no longer detected 16–24 h after removal of fluorescent crotamine from the culture medium. Parallel experiments demonstrated that Cy3-conjugated crotamine was efficiently internalized at concentrations as low as 10 nM at 37°C but not at 4°C.
3. Nuclear localization of crotamine
Cy3-crotamine was visualized in the nuclear and perinuclear regions in different cell types from bone marrow and peritoneal liquid cells (Fig. 1G-I
, Fig. 2
A–C; G–S). DNA DAPI staining partially overlapped crotamine localization in the nuclei.
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Injection of unlabeled crotamine was followed by injection of Cy3-crotamine; after another 15 min fluorescence was seen to be restricted to the cytoplasm, preferential to the perinuclear space, indicating a saturation of binding sites by unlabeled crotamine within the nucleus. The same results were observed in human fibroblast by treating cells with unlabeled and Cy3-crotamine.
4. Crotamine as a marker of centrioles and during the cell cycle
Cy3-crotamine also seems to function as a marker of the centrosome cycle. Figure 2A
shows centrioles from peritoneal liquid cells labeled with Cy3-crotamine at different phases of the cell cycle. Anti-
-tubulin antibody was used in ES cells to highlight the spindle and esters, small star-like structures formed by new microtubules growing out from centrosomes during mitosis (Fig. 2B
). Cy3-crotamine colocalizes with esters immunostained by anti-tubulin, indicating its centrosome association (Fig. 2C
). Cy3-crotamine labels the chromosomes attached to the -tubulin immunostained spindle (Fig. 2C
).
Centrosome duplication and separation were observed in Cy3-crotamine-treated peritoneal liquid cells (Fig. 2D-S
). Centrioles and the associated centrosome matrix were initially observed as a single complex (Fig. 2D-F
). In S/G2, two centrioles could be seen as they began to move apart (Fig. 2G-L
). At metaphase (Fig. 2M-O
) and anaphase (Fig. 2P-S
), the centrioles were observed in opposite poles. In S/G2 phase, the fluorescent signal appeared on the chromosomes (Fig. 2G-I
) and became very strong at metaphase (Fig. 2M-O
).
5. Crotamine as a marker of actively proliferating cells
Cy3-crotamine cell labeling varied in intensity (Fig. 1A-E
). This was striking when ES cells were induced to differentiate through Ebs: a 6 day monolayer culture of a differentiating Eb was treated with Cy3-crotamine for 24 h, and undifferentiated ES cells within the Eb, which supposedly were actively proliferating, appeared strongly labeled.
A 5-BrdU proliferation assay was used to investigate whether crotamine preferentially labels actively proliferating cells in differentiating Ebs. 5-BrdU incorporation into DNA during replication was monitored by anti-BrdU antibody immunostaining. Cy3-crotamine was added with 5-BrdU into cultures of differentiating Ebs. While an intensive crotamine labeling was observed in the areas of actively proliferating ES cells, a weak Cy3-fluorescence was seen in areas of predominantly nondividing cells. Crotamine and 5-BrdU had nuclear localization in the actively proliferating cells, but overlapping was not complete, indicating different sites of interaction of crotamine and 5-BrdU.
CONCLUSION AND SIGNIFICANCE
ES cells were used to study the toxicity of crotamine, a toxin from rattlesnake venom. Crotamine in micromolar concentrations had no toxic effect on ES cells in vitro. We demonstrated it did not inhibit Ebs formation or ES cell differentiation into contracting myocardium and was nontoxic for developing mouse blastocysts.
We investigated crotamine internalization in vitro in different cell lines and in vivo in cell types isolated from mouse. Internalization was always effective and cells showed strong labeling within 5 min of treatment with Cy3-crotamine, a feature shared with other cell-penetrating proteins. Signals of similar intensity were observed in cells incubated with 1 µM Cy3-crotamine for 1–48 h. Cells incubated with Cy3-crotamine at concentrations as low as 10 nM were rapidly and intensively labeled.
Once internalized, Cy3-crotamine was observed in the perinuclear space and cell nucleus. Its nuclear localization was confirmed by DAPI staining and a peptide competition assay demonstrating that Cy3-crotamine was not translocated into the nucleus when binding sites had been saturated by unlabeled crotamine. Since fixation is known to produce artificial nuclear localization of cationic peptides, crotamine penetration into the nuclei was studied in unfixed and fixed cells; its nuclear localization was observed in both cases.
Crotamine was shown to bind to DAPI-stained and 5-BrdU-labeled metaphase chromosomes. It seems to bind to chromosomes in S/G2 and G2/M phases, when fluorescent labeling becomes evident on all chromosomes. At the end of telophase, crotamine is restricted to the cytoplasm. After 16–24 h of its removal from cell culture medium, crotamine is not detected in cells. Crotamine differs in this regard from herpes simplex VP22 protein, which binds to chromatin after internalization and segregates to daughter cells.
Crotamine was demonstrated to specifically associate with centrosomes by simultaneous anti-tubulin immunostaining and crotamine labeling of esters, allowing the position of the cell in the cell cycle to be assigned. As a centriolar marker, it appears to be a tool to monitor unfixed human tumor cells characterized by an abnormally high number of centrosomes. Crotamine was shown to selectively label actively proliferating living cells both in vivo and in vitro (Fig. 3
).
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Crotamine was characterized as a cell-penetrating protein with nuclear localization in vitro and in vivo. The nature of crotamine interaction with chromatin is unclear, but our morphological data indicate that the mechanisms involved differ from those of DAPI or 5-BrdU.
FOOTNOTES
2 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1459fje; doi: 10.1096/fj.03-1459fje
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