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Disruption and de novo formation of nanotubular membrane extensions in SW620 colon carcinoma cell line during cell division
Cell Biology International 29 (2005) 929e931 www.elsevier.com/locate/cellbi
Disruption and de novo formation of nanotubular membrane extensions in SW620 colon carcinoma cell line during cell division Mauro A.A. Castro a,b,c, Veroˆnica A. Grieneisen a, Rita M.C. de Almeida a,* a
Instituto de Fı´sica, Universidade Federal do Rio Grande do Sul, Avenue Bento Gonc¸alves 9500, C.P. 15051, Porto Alegre 91501-970, Brazil b Departamento de Bioquı´mica, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos 2600 e anexo, Porto Alegre 90035-003, Brazil c Universidade Luterana do Brasil, Rua Miguel Tostes 101, Canoas 92420-280, Brazil Received 24 November 2004; revised 11 April 2005; accepted 23 May 2005
Abstract A novel biological principle of cell-to-cell interaction based on membrane continuity of nanotubular channels has recently been described. These contacts are extremely dynamic and sensitive to mechanical stress, which causes their rapid breakage and retraction. Here we demonstrate that functional mechanical stress generated during cell division can disrupt membrane nanotubes, which are formed de novo when filopodia-like projections on one cell make contact with a neighbouring cell, using the SW620 colon carcinoma cell line. Considering the general principal of decreasing cellecell interactions during tumour progression, our observation is appealing because this new phenomenon may be valid for neoplastic cells. Ó 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Intercellular contact; Cancer cells; Membrane protrusion; Tunnelling nanotubes; Video microscopy
1. Introduction Cell-to-cell communication is achieved by a variety of means, from diffusive signals and gap junctions in animals to direct cytoplasmic connections called plasmodesmata in higher plants (Green and Watt, 2004). Although plasmodesmata have long been appreciated in plants, a similar type of intercellular communication has only recently been discovered in animal cells, known as tunnelling nanotubes (TNTs), which are formed through cytoplasmic protrusions. They were first observed in cultures of pheochromocytoma (PC12) cells (Rustom et al., 2004; Cilia and Jackson, 2004). Cytoplasmic protrusions are an early step in several cellular processes, including neurite outgrowth, phagocytosis, and formation of cellecell junctions and cell migration, which plays
* Corresponding author. E-mail address: [email protected] (R.M.C. de Almeida).
a central role in both normal and pathological processes, including embryonic development, wound healing, inflammation, and tumour metastasis. Several types of cytoplasmic protrusion have been described and these include lamellipodia, pseudopodia and filopodia (Rorth, 2003). TNTs are ultrafine cell-to-cell membrane extensions, whose dimensions are similar to filopodia but anchor to adjacent cells (Baluska et al., 2004). They contain F-actin that is fused to adjacent cells to form actin-based cell-to-cell channels of 50e200 nm wide and several cell diameters long. This physical link allows organelles to be unidirectionally transferred to the target cell by an actin-mediated mechanism (Rustom et al., 2004). It represents a new long-range form of cell communication, probably having an important role in the regulation of a diverse range of cellular processes through the transport of endosomal-like vesicles towards adjacent cells (Croager, 2004). It is possible that these cytoplasmic protrusions are related to Drosophila imaginal disc cells that make polarized actin-based extensions (cytonemes) and may transmit signals between
1065-6995/$ - see front matter Ó 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2005.05.013
M.A.A. Castro et al. / Cell Biology International 29 (2005) 929e931
930
outlying cells (Ramirez-Weber and Kornberg, 2000), but the degree to which apparently similar structures and processes are homologous is unclear. To date, only a small number of cell types have been shown to produce these intercellular adhesion structures. With time-lapse video microscopy and image analysis to examine cell-to-cell contact dynamics, we show evidence that TNTs are disrupted and formed de novo in the SW620 colon carcinoma cell line following mechanical stress due to cell division.
microscope equipped with a black and white CCD camera and a heating chamber fixed on the microscope stage (thermo-sensor electronically controlled at 37 G 0.1 C). Images were processed using ImageJ 1.32j software (PC version for NIH-image), optimized by adjusting contrast, grey scale and saved in a picture file format before being analysed. The frames were captured at 3-min intervals for 12 h at 640 ! 480 pixels; images in Fig. 1 correspond to 320 ! 210 pixels of the original video frames (Foghsgaard et al., 2001; Tibaldi et al., 2002).
2. Material and methods
Using time-lapse video microscopy, we observed two adjacent cells initially connected by long membrane protrusions, producing two adhesion-free perpendicularly connected nanotubes (Fig. 1a). These nanotubes establish a belt-like system through which one cell is anchored and pulls the main axis of the protrusion, which is attached on a focal point at its end (Fig. 1b and c). The membrane protrusions are stretched further due to cell movements until one cell enters mitosis, detaches from substrata and loses the membrane contacts (Fig. 1deg). After filopodia produced by the daughter cell reaches the former main axis protrusion (Fig. 1hej), a new contact is made, establishing the belt-like system de novo. This observation shows that functional mechanical stress generated during cell division can cause rapid breakage and retraction of connected nanotubes, which can also be reformed when filopodia-like projections on one cell make contact with a neighbouring cell. Given that disruption of cellecell junctions and adhesion is a general principal for neoplastic cells (Guo and Giancotti, 2004; Friedl and Wolf, 2003), membrane nanotubes may add new insights into the cellular basis of carcinogenesis by directly or indirectly interfering with cellecell signalling. It may be
3. Results and discussion
2.1. Cell culture The human colon carcinoma cell line SW620 was obtained from the American Type Culture Collection (Rockville, MD, USA; ATCC number CCL 227; colon adenocarcinoma, lymph node metastasis, passage no. 83). The cells were grown as an adherent monolayer in 25 cm2 culture flasks at 37 C in a 5% CO2 humidified atmosphere and maintained in RPMI 1640 medium supplemented with 10% FCS (v/v). Trypsin treatment was carried out at 37 C for 2 min with a mixture of 0.05% (w/v) trypsin and 0.02% (w/v) EDTA. After trypsinization, cells were counted in a hemocytometer chamber, diluted to appropriate numbers and seeded (Castro et al., 2003).
2.2. Time-lapse video microscopy Cells were seeded onto 40 mm diameter circular cell culture vessels with their surface modified for optimal cell attachment (CorningÒ Cell Culture Polystyrene) and allowed to grow to 40% confluence. The culture medium (RPMI 1640) was changed to CO2-independent pH buffering medium (Gibco BRL) supplemented with 10% serum and L-gln to a final concentration of 2 mM. Culture vessels were then filled with medium, tightened and sealed with parafilm to prevent evaporation. Phase contrast time-lapse photographs were obtained at 200! magnification using an Olympus-IX81 inverted
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Fig. 1. Cell-to-cell contact dynamics in SW620 colon carcinoma cell line. (aej) Select frames of a phase contrast video microscopy sequence acquired at 3-min intervals over 12 h. Numbers refer to corresponding cells. One protrusion is attached at one adhesion point (white arrows) and also contacts a neighbouring cell (black arrows). As the neighbouring cell detaches due to mitosis (d), the protrusion is pulled and stretches until the contact is lost (e and f). New cells are formed after cytokinesis (g and h) and one daughter cell makes a new contact with the same protrusion (i and j; arrowhead). Scale bar, 25 mm.
M.A.A. Castro et al. / Cell Biology International 29 (2005) 929e931
that these cytoplasm protrusions will ultimately be found in all cell types, but it will be important to verify the existence and role of TNTs in more cancer models and in other contexts before concluding that they constitute a widespread phenomenon. Future studies will uncover the full complexity and relevance of these membrane contacts for cancer cells, and it may well become apparent that some changes are more important than others. Acknowledgements This study was supported by CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico e DF, Brazil). References Baluska F, Hlavacka A, Volkmann D, Menzel D. Getting connected: actinbased cell-to-cell channels in plants and animals. Trends Cell Biol 2004;14:404e8. Castro MAA, Klamt F, Grieneisen VA, Grivicich I, Moreira JCF. Gompertzian growth pattern correlated with phenotypic organization of colon carcino-
931
ma, malignant glioma and non-small cell lung carcinoma cell lines. Cell Prolif 2003;36:65e73. Cilia ML, Jackson D. Plasmodesmata form and function. Curr Opin Cell Biol 2004;16:500e6. Croager E. Cellecell communication e opening communication channels. Nat Rev Mol Cell Biol 2004;5:252. Foghsgaard L, Wissing D, Mauch D, Lademann U, Bastholm L, Boes M, et al. Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor. J Cell Biol 2001;153:999e1009. Friedl P, Wolf K. Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer 2003;3:362e74. Green K, Watt F. Cell-to-cell contact and extracellular matrix. Curr Opin Cell Biol 2004;16:465e9. Guo WJ, Giancotti FG. Integrin signalling during tumour progression. Nat Rev Mol Cell Biol 2004;5:816e26. Ramirez-Weber F, Kornberg TB. Signaling reaches to new dimensions in Drosophila imaginal discs. Cell 2000;103:189e92. Rorth P. Communication by touch: role of cellular extensions in complex animals. Cell 2003;112:595e8. Rustom A, Saffrich R, Markovic I, Walther PL, Gerdes HH. Nanotubular highways for intercellular organelle transport. Science 2004;303: 1007e10. Tibaldi EV, Salgia R, Reinherz EL. CD2 molecules redistribute to the uropod during T cell scanning: implications for cellular activation and immune surveillance. Proc Natl Acad Sci U S A 2002;99:7582e7.
Disruption and de novo formation of nanotubular membrane extensions in SW620 colon carcinoma cell line during cell division Mauro A.A. Castro a,b,c, Veroˆnica A. Grieneisen a, Rita M.C. de Almeida a,* a
Instituto de Fı´sica, Universidade Federal do Rio Grande do Sul, Avenue Bento Gonc¸alves 9500, C.P. 15051, Porto Alegre 91501-970, Brazil b Departamento de Bioquı´mica, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos 2600 e anexo, Porto Alegre 90035-003, Brazil c Universidade Luterana do Brasil, Rua Miguel Tostes 101, Canoas 92420-280, Brazil Received 24 November 2004; revised 11 April 2005; accepted 23 May 2005
Abstract A novel biological principle of cell-to-cell interaction based on membrane continuity of nanotubular channels has recently been described. These contacts are extremely dynamic and sensitive to mechanical stress, which causes their rapid breakage and retraction. Here we demonstrate that functional mechanical stress generated during cell division can disrupt membrane nanotubes, which are formed de novo when filopodia-like projections on one cell make contact with a neighbouring cell, using the SW620 colon carcinoma cell line. Considering the general principal of decreasing cellecell interactions during tumour progression, our observation is appealing because this new phenomenon may be valid for neoplastic cells. Ó 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Intercellular contact; Cancer cells; Membrane protrusion; Tunnelling nanotubes; Video microscopy
1. Introduction Cell-to-cell communication is achieved by a variety of means, from diffusive signals and gap junctions in animals to direct cytoplasmic connections called plasmodesmata in higher plants (Green and Watt, 2004). Although plasmodesmata have long been appreciated in plants, a similar type of intercellular communication has only recently been discovered in animal cells, known as tunnelling nanotubes (TNTs), which are formed through cytoplasmic protrusions. They were first observed in cultures of pheochromocytoma (PC12) cells (Rustom et al., 2004; Cilia and Jackson, 2004). Cytoplasmic protrusions are an early step in several cellular processes, including neurite outgrowth, phagocytosis, and formation of cellecell junctions and cell migration, which plays
* Corresponding author. E-mail address: [email protected] (R.M.C. de Almeida).
a central role in both normal and pathological processes, including embryonic development, wound healing, inflammation, and tumour metastasis. Several types of cytoplasmic protrusion have been described and these include lamellipodia, pseudopodia and filopodia (Rorth, 2003). TNTs are ultrafine cell-to-cell membrane extensions, whose dimensions are similar to filopodia but anchor to adjacent cells (Baluska et al., 2004). They contain F-actin that is fused to adjacent cells to form actin-based cell-to-cell channels of 50e200 nm wide and several cell diameters long. This physical link allows organelles to be unidirectionally transferred to the target cell by an actin-mediated mechanism (Rustom et al., 2004). It represents a new long-range form of cell communication, probably having an important role in the regulation of a diverse range of cellular processes through the transport of endosomal-like vesicles towards adjacent cells (Croager, 2004). It is possible that these cytoplasmic protrusions are related to Drosophila imaginal disc cells that make polarized actin-based extensions (cytonemes) and may transmit signals between
1065-6995/$ - see front matter Ó 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2005.05.013
M.A.A. Castro et al. / Cell Biology International 29 (2005) 929e931
930
outlying cells (Ramirez-Weber and Kornberg, 2000), but the degree to which apparently similar structures and processes are homologous is unclear. To date, only a small number of cell types have been shown to produce these intercellular adhesion structures. With time-lapse video microscopy and image analysis to examine cell-to-cell contact dynamics, we show evidence that TNTs are disrupted and formed de novo in the SW620 colon carcinoma cell line following mechanical stress due to cell division.
microscope equipped with a black and white CCD camera and a heating chamber fixed on the microscope stage (thermo-sensor electronically controlled at 37 G 0.1 C). Images were processed using ImageJ 1.32j software (PC version for NIH-image), optimized by adjusting contrast, grey scale and saved in a picture file format before being analysed. The frames were captured at 3-min intervals for 12 h at 640 ! 480 pixels; images in Fig. 1 correspond to 320 ! 210 pixels of the original video frames (Foghsgaard et al., 2001; Tibaldi et al., 2002).
2. Material and methods
Using time-lapse video microscopy, we observed two adjacent cells initially connected by long membrane protrusions, producing two adhesion-free perpendicularly connected nanotubes (Fig. 1a). These nanotubes establish a belt-like system through which one cell is anchored and pulls the main axis of the protrusion, which is attached on a focal point at its end (Fig. 1b and c). The membrane protrusions are stretched further due to cell movements until one cell enters mitosis, detaches from substrata and loses the membrane contacts (Fig. 1deg). After filopodia produced by the daughter cell reaches the former main axis protrusion (Fig. 1hej), a new contact is made, establishing the belt-like system de novo. This observation shows that functional mechanical stress generated during cell division can cause rapid breakage and retraction of connected nanotubes, which can also be reformed when filopodia-like projections on one cell make contact with a neighbouring cell. Given that disruption of cellecell junctions and adhesion is a general principal for neoplastic cells (Guo and Giancotti, 2004; Friedl and Wolf, 2003), membrane nanotubes may add new insights into the cellular basis of carcinogenesis by directly or indirectly interfering with cellecell signalling. It may be
3. Results and discussion
2.1. Cell culture The human colon carcinoma cell line SW620 was obtained from the American Type Culture Collection (Rockville, MD, USA; ATCC number CCL 227; colon adenocarcinoma, lymph node metastasis, passage no. 83). The cells were grown as an adherent monolayer in 25 cm2 culture flasks at 37 C in a 5% CO2 humidified atmosphere and maintained in RPMI 1640 medium supplemented with 10% FCS (v/v). Trypsin treatment was carried out at 37 C for 2 min with a mixture of 0.05% (w/v) trypsin and 0.02% (w/v) EDTA. After trypsinization, cells were counted in a hemocytometer chamber, diluted to appropriate numbers and seeded (Castro et al., 2003).
2.2. Time-lapse video microscopy Cells were seeded onto 40 mm diameter circular cell culture vessels with their surface modified for optimal cell attachment (CorningÒ Cell Culture Polystyrene) and allowed to grow to 40% confluence. The culture medium (RPMI 1640) was changed to CO2-independent pH buffering medium (Gibco BRL) supplemented with 10% serum and L-gln to a final concentration of 2 mM. Culture vessels were then filled with medium, tightened and sealed with parafilm to prevent evaporation. Phase contrast time-lapse photographs were obtained at 200! magnification using an Olympus-IX81 inverted
0 min
a
192 min
b
1
1
2
396 min
f
1
2
471 min
g
483 min
1
558 min
i
1
3 4
2
2
2
h
390 min
e
1
1
3 4
384 min
d
1
1
2
282 min
c
672 min
j
1
3 4
3 4
Fig. 1. Cell-to-cell contact dynamics in SW620 colon carcinoma cell line. (aej) Select frames of a phase contrast video microscopy sequence acquired at 3-min intervals over 12 h. Numbers refer to corresponding cells. One protrusion is attached at one adhesion point (white arrows) and also contacts a neighbouring cell (black arrows). As the neighbouring cell detaches due to mitosis (d), the protrusion is pulled and stretches until the contact is lost (e and f). New cells are formed after cytokinesis (g and h) and one daughter cell makes a new contact with the same protrusion (i and j; arrowhead). Scale bar, 25 mm.
M.A.A. Castro et al. / Cell Biology International 29 (2005) 929e931
that these cytoplasm protrusions will ultimately be found in all cell types, but it will be important to verify the existence and role of TNTs in more cancer models and in other contexts before concluding that they constitute a widespread phenomenon. Future studies will uncover the full complexity and relevance of these membrane contacts for cancer cells, and it may well become apparent that some changes are more important than others. Acknowledgements This study was supported by CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico e DF, Brazil). References Baluska F, Hlavacka A, Volkmann D, Menzel D. Getting connected: actinbased cell-to-cell channels in plants and animals. Trends Cell Biol 2004;14:404e8. Castro MAA, Klamt F, Grieneisen VA, Grivicich I, Moreira JCF. Gompertzian growth pattern correlated with phenotypic organization of colon carcino-
931
ma, malignant glioma and non-small cell lung carcinoma cell lines. Cell Prolif 2003;36:65e73. Cilia ML, Jackson D. Plasmodesmata form and function. Curr Opin Cell Biol 2004;16:500e6. Croager E. Cellecell communication e opening communication channels. Nat Rev Mol Cell Biol 2004;5:252. Foghsgaard L, Wissing D, Mauch D, Lademann U, Bastholm L, Boes M, et al. Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor. J Cell Biol 2001;153:999e1009. Friedl P, Wolf K. Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer 2003;3:362e74. Green K, Watt F. Cell-to-cell contact and extracellular matrix. Curr Opin Cell Biol 2004;16:465e9. Guo WJ, Giancotti FG. Integrin signalling during tumour progression. Nat Rev Mol Cell Biol 2004;5:816e26. Ramirez-Weber F, Kornberg TB. Signaling reaches to new dimensions in Drosophila imaginal discs. Cell 2000;103:189e92. Rorth P. Communication by touch: role of cellular extensions in complex animals. Cell 2003;112:595e8. Rustom A, Saffrich R, Markovic I, Walther PL, Gerdes HH. Nanotubular highways for intercellular organelle transport. Science 2004;303: 1007e10. Tibaldi EV, Salgia R, Reinherz EL. CD2 molecules redistribute to the uropod during T cell scanning: implications for cellular activation and immune surveillance. Proc Natl Acad Sci U S A 2002;99:7582e7.