Limits...
Partitioning and Exocytosis of Secretory Granules during Division of PC12 Cells.

Bukoreshtliev NV, Hodneland E, Eichler TW, Eifart P, Rustom A, Gerdes HH - Int J Cell Biol (2012)

Bottom Line: By combining ultrastructural analyses and time-lapse microscopy, we here show that, in dividing PC12 cells, the prominent peripheral localization of secretory granules is retained during prophase but clearly reduced during prometaphase, ending up with only few peripherally localized secretory granules in metaphase cells.During anaphase and telophase, secretory granules exhibited a pronounced movement towards the cell midzone and, evidently, their tracks colocalized with spindle microtubules.During cytokinesis, secretory granules were excluded from the midbody and accumulated at the bases of the intercellular bridge.

View Article: PubMed Central - PubMed

Affiliation: Interdisciplinary Center for Neurosciences (IZN), Department of Neurobiology, University of Heidelberg, INF 364, 69120 Heidelberg, Germany.

ABSTRACT
The biogenesis, maturation, and exocytosis of secretory granules in interphase cells have been well documented, whereas the distribution and exocytosis of these hormone-storing organelles during cell division have received little attention. By combining ultrastructural analyses and time-lapse microscopy, we here show that, in dividing PC12 cells, the prominent peripheral localization of secretory granules is retained during prophase but clearly reduced during prometaphase, ending up with only few peripherally localized secretory granules in metaphase cells. During anaphase and telophase, secretory granules exhibited a pronounced movement towards the cell midzone and, evidently, their tracks colocalized with spindle microtubules. During cytokinesis, secretory granules were excluded from the midbody and accumulated at the bases of the intercellular bridge. Furthermore, by measuring exocytosis at the single granule level, we showed, that during all stages of cell division, secretory granules were competent for regulated exocytosis. In conclusion, our data shed new light on the complex molecular machinery of secretory granule redistribution during cell division, which facilitates their release from the F-actin-rich cortex and active transport along spindle microtubules.

No MeSH data available.


Quantification and comparison of the intensity of SgII surface staining of interphase and metaphase cells. PC12 cells were stained with WGA (A), Hoechst (B) and by immunocytochemistry for surface-associated SgII (E). Images displaying the WGA stain were used for segmentation (C). Asterisk in (C) indicates cell borders that are not detected. The Hoechst stain was used for the identification of metaphase and interphase cells (B). A mask was constructed for the quantification of the surface-associated SgII signal (red double-line in (F)) delineating the PM of PC12 cells located at the outside of the cell cluster. For more details please see Materials and Methods. Scale bar, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3375158&req=5

fig10: Quantification and comparison of the intensity of SgII surface staining of interphase and metaphase cells. PC12 cells were stained with WGA (A), Hoechst (B) and by immunocytochemistry for surface-associated SgII (E). Images displaying the WGA stain were used for segmentation (C). Asterisk in (C) indicates cell borders that are not detected. The Hoechst stain was used for the identification of metaphase and interphase cells (B). A mask was constructed for the quantification of the surface-associated SgII signal (red double-line in (F)) delineating the PM of PC12 cells located at the outside of the cell cluster. For more details please see Materials and Methods. Scale bar, 10 μm.

Mentions: Under control conditions, SgII was barely detectable at the surface of both interphase and mitotic PC12 cells (Figure 9(B)). However, stimulated cells exhibited a prominent surface staining for SgII (Figure 9(A)). Interestingly, also mitotic cells responded to the stimulus and frequently displayed newly exocytosed SgII at the cell surface (Figures 9(E), 9(G), 9(I)). It is of note that for both interphase and mitotic cells the surface signal intensity varied considerably from cell to cell (Figure 9(A)). For a quantitative and unbiased evaluation of the fraction of surface-stained cells as well as the intensity of the surface stain for both interphase and metaphase cells, we designed a semiautomatic algorithm for the calculation of the surface-staining signal intensity of user-selected cells (for details please see Figure 10 and Materials and Methods). This quantification revealed that approximately 80% of interphase and 65% of metaphase cells displayed signal intensities above background (Figure 9(K)). Furthermore, the average signal intensity of the surface-stain for metaphase cells was approximately 60% as compared to that of interphase cells (Figure 9(K)). Taken together, these quantitative data show that, although with an approximately 2-fold reduced efficiency, dividing cells are competent for regulated exocytosis.


Partitioning and Exocytosis of Secretory Granules during Division of PC12 Cells.

Bukoreshtliev NV, Hodneland E, Eichler TW, Eifart P, Rustom A, Gerdes HH - Int J Cell Biol (2012)

Quantification and comparison of the intensity of SgII surface staining of interphase and metaphase cells. PC12 cells were stained with WGA (A), Hoechst (B) and by immunocytochemistry for surface-associated SgII (E). Images displaying the WGA stain were used for segmentation (C). Asterisk in (C) indicates cell borders that are not detected. The Hoechst stain was used for the identification of metaphase and interphase cells (B). A mask was constructed for the quantification of the surface-associated SgII signal (red double-line in (F)) delineating the PM of PC12 cells located at the outside of the cell cluster. For more details please see Materials and Methods. Scale bar, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375158&req=5

fig10: Quantification and comparison of the intensity of SgII surface staining of interphase and metaphase cells. PC12 cells were stained with WGA (A), Hoechst (B) and by immunocytochemistry for surface-associated SgII (E). Images displaying the WGA stain were used for segmentation (C). Asterisk in (C) indicates cell borders that are not detected. The Hoechst stain was used for the identification of metaphase and interphase cells (B). A mask was constructed for the quantification of the surface-associated SgII signal (red double-line in (F)) delineating the PM of PC12 cells located at the outside of the cell cluster. For more details please see Materials and Methods. Scale bar, 10 μm.
Mentions: Under control conditions, SgII was barely detectable at the surface of both interphase and mitotic PC12 cells (Figure 9(B)). However, stimulated cells exhibited a prominent surface staining for SgII (Figure 9(A)). Interestingly, also mitotic cells responded to the stimulus and frequently displayed newly exocytosed SgII at the cell surface (Figures 9(E), 9(G), 9(I)). It is of note that for both interphase and mitotic cells the surface signal intensity varied considerably from cell to cell (Figure 9(A)). For a quantitative and unbiased evaluation of the fraction of surface-stained cells as well as the intensity of the surface stain for both interphase and metaphase cells, we designed a semiautomatic algorithm for the calculation of the surface-staining signal intensity of user-selected cells (for details please see Figure 10 and Materials and Methods). This quantification revealed that approximately 80% of interphase and 65% of metaphase cells displayed signal intensities above background (Figure 9(K)). Furthermore, the average signal intensity of the surface-stain for metaphase cells was approximately 60% as compared to that of interphase cells (Figure 9(K)). Taken together, these quantitative data show that, although with an approximately 2-fold reduced efficiency, dividing cells are competent for regulated exocytosis.

Bottom Line: By combining ultrastructural analyses and time-lapse microscopy, we here show that, in dividing PC12 cells, the prominent peripheral localization of secretory granules is retained during prophase but clearly reduced during prometaphase, ending up with only few peripherally localized secretory granules in metaphase cells.During anaphase and telophase, secretory granules exhibited a pronounced movement towards the cell midzone and, evidently, their tracks colocalized with spindle microtubules.During cytokinesis, secretory granules were excluded from the midbody and accumulated at the bases of the intercellular bridge.

View Article: PubMed Central - PubMed

Affiliation: Interdisciplinary Center for Neurosciences (IZN), Department of Neurobiology, University of Heidelberg, INF 364, 69120 Heidelberg, Germany.

ABSTRACT
The biogenesis, maturation, and exocytosis of secretory granules in interphase cells have been well documented, whereas the distribution and exocytosis of these hormone-storing organelles during cell division have received little attention. By combining ultrastructural analyses and time-lapse microscopy, we here show that, in dividing PC12 cells, the prominent peripheral localization of secretory granules is retained during prophase but clearly reduced during prometaphase, ending up with only few peripherally localized secretory granules in metaphase cells. During anaphase and telophase, secretory granules exhibited a pronounced movement towards the cell midzone and, evidently, their tracks colocalized with spindle microtubules. During cytokinesis, secretory granules were excluded from the midbody and accumulated at the bases of the intercellular bridge. Furthermore, by measuring exocytosis at the single granule level, we showed, that during all stages of cell division, secretory granules were competent for regulated exocytosis. In conclusion, our data shed new light on the complex molecular machinery of secretory granule redistribution during cell division, which facilitates their release from the F-actin-rich cortex and active transport along spindle microtubules.

No MeSH data available.