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Loose and compact agglomerates of 50 nm microvesicles derived from Golgi and endoplasmic reticulum membranes in pre- and in -apoptotic Mycoplasma infected HeLa cells: host-parasite interactions under the transmission electron microscope.

Sesso A, Yamashiro-Kanashiro EH, Orii NM, Taniwaki NN, Kawakami J, Carneiro SM - Rev. Inst. Med. Trop. Sao Paulo (2015 Jan-Feb)

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Affiliation: Laboratório de Imunopatologia, Instituto de Medicina Tropical (IMT) de São Paulo.

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São Paulo, November 17, 2014 Dear Editor The fine structure of apoptotic HeLa cells from cultures contaminated withmycoplasma in early and in advanced stages of the cell demise process differs from those sofar described in apoptotic cells... These attain sizes larger(Fig. 2) than those of the clusters of microvesicles derived from the fragmentation ofGolgi saccules seen in mitotic (LUCOCQ et al., 1989; SESSO etal., 1999) and in apoptotic (SESSO et al., 1999) cells.Contemporaneously two major cytoplasmic alterations may be noted in contaminated cellsnamely when treated with staurosporine... All cytoplasmic membrane bound organelles as peroxisomes, lysosome-endosomes andthe Golgi apparatus derive from microvesicles that bud off from the ER... The ER is alsomobilized by promoters of cellular stress (references in DOLAI & ADAK, 2014)... The heredescribed structural deviation of the ER-Golgi interface from the normal condition mayrepresent more than only a mycoplasma induced alteration of the programmed cell deathmechanism... It is speculative, whether this initial accumulation of microvesicles in cellswith typical normal nuclei is part of a general forewarning mechanism of cell defence... In aless intense cell stress than that occurring here the observed changes of the TER couldeventually pass undetected under the transmission electron microscope... The mycoplasmas fine structures of our samples are identical (NIR-PAZ etal., 2002; KORNSPAN et al., 2010;) and similar (EDWARDS &FOGH, 1960; HUMMELER et al., 1965; TAYLOR-ROBINSON etal., 1991) to those from various mycoplasmas strains seen in cultures and ininfected cells.

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7 are a synopsis of fine structural observations in some 50 samplesof staurosporine (0.1 µM - 2.0 µM for 2 - 24h) and 10 control samples of HeLa cellcultures contaminated with mycoplasma. Fig.1 is from an apparent normal cell at low microscopic exam. Figs 2, 6 and7 and 3 and 4 are from apoptotic cells exposed to staurosporine 0.5 µM/3h and 0.1µM/6h, respectively. Fig. 5 is from a non apoptotic cell exposed to staurosporine0.1 µM/4h. Bars -1 µM. Fig. 1 - HeLa cell-from a heavily contaminated cell culture devoid ofstaurosporine where apoptosis occurred. Normal nucleus (N). Smooth, branched ERprofiles (extremities of thin white lines departing from the central whiteasterisks in the area delimited by the lower (L) dotted line. Conglomerates of 50nm (lower and upper right part of the figure). Part of the microvesicles bud offfrom smooth ER membranes (arrow head) and from tubular elements (small arrows).The majority of the grouped microvesicles pinch off from the extremities ofstacked Golgi saccules (thin arrow heads at the periphery of the frontally exposeddense ellipsoidal-like profile delimited by U)]. Stacked, thin Golgi saccularprofiles of various lengths that often appear dense ( ] ). Fig. 2 -Large, compact conglomeration of various sized microvesicles with predominance ofthe ones with 50 nm. Groups of former dilated stacked Golgi saccules (blackasterisks) are interspersed among the microvesicles. A large such dilated sacculecontains remnants of a parasite or of material of parasitic origin (MPO) (emptytriangle). Fig. 3 - The cytoplasm of this apoptotic cell is delimitedin two concentric major regions. The peripheral one is partitioned into adjacentblebs containing predominantly membranes of the ER. The inner part of thecytoplasm contains clustered, swollen mitochondria. Fig. 4 -Dismantling of the peripheral apoptotic cytoplasm by a contemporaneous detachmentof the previously formed bleb regions. Fig. 5 - Non apoptoticinfected (cysts of parasitic origin, upper arrows) cell, with an abnormallyelongated thin cytoplasm. The free villus-like structures often appear curvedaside spheroidal 50-100 nm (thin arrow) elements. They assemble in various degreesin the sectors of the contaminated cells apoptotic or not undergoing progressivedismantling of the peripheral cytoplasm. Fig. 6 - Most of thecytoplasm above the apoptotic nucleus is occupied by membrane bound void spaces ofvarious sizes and forms. The mitochondria are unusually dense, a common occurrencein contaminated cells, apoptotic or not. Adherent to the limiting membrane of thespace indicated (open triangle) a fluffy material possibly derived from MPO.Fig. 7 - Sector of an apoptotic cytoplasm with net-like membranalarrangement as in Fig. 6. Compact mass of parasitic material in a cystic-like form(open triangle). The upper, middle and vertical white lines indicate remnants ofparasitic origin that were not completely removed by the processing of the cells.Part of an elongate membrane bound space possibly still fully occupied by MPO isindicated by the lower line that branches in two.
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f01: 7 are a synopsis of fine structural observations in some 50 samplesof staurosporine (0.1 µM - 2.0 µM for 2 - 24h) and 10 control samples of HeLa cellcultures contaminated with mycoplasma. Fig.1 is from an apparent normal cell at low microscopic exam. Figs 2, 6 and7 and 3 and 4 are from apoptotic cells exposed to staurosporine 0.5 µM/3h and 0.1µM/6h, respectively. Fig. 5 is from a non apoptotic cell exposed to staurosporine0.1 µM/4h. Bars -1 µM. Fig. 1 - HeLa cell-from a heavily contaminated cell culture devoid ofstaurosporine where apoptosis occurred. Normal nucleus (N). Smooth, branched ERprofiles (extremities of thin white lines departing from the central whiteasterisks in the area delimited by the lower (L) dotted line. Conglomerates of 50nm (lower and upper right part of the figure). Part of the microvesicles bud offfrom smooth ER membranes (arrow head) and from tubular elements (small arrows).The majority of the grouped microvesicles pinch off from the extremities ofstacked Golgi saccules (thin arrow heads at the periphery of the frontally exposeddense ellipsoidal-like profile delimited by U)]. Stacked, thin Golgi saccularprofiles of various lengths that often appear dense ( ] ). Fig. 2 -Large, compact conglomeration of various sized microvesicles with predominance ofthe ones with 50 nm. Groups of former dilated stacked Golgi saccules (blackasterisks) are interspersed among the microvesicles. A large such dilated sacculecontains remnants of a parasite or of material of parasitic origin (MPO) (emptytriangle). Fig. 3 - The cytoplasm of this apoptotic cell is delimitedin two concentric major regions. The peripheral one is partitioned into adjacentblebs containing predominantly membranes of the ER. The inner part of thecytoplasm contains clustered, swollen mitochondria. Fig. 4 -Dismantling of the peripheral apoptotic cytoplasm by a contemporaneous detachmentof the previously formed bleb regions. Fig. 5 - Non apoptoticinfected (cysts of parasitic origin, upper arrows) cell, with an abnormallyelongated thin cytoplasm. The free villus-like structures often appear curvedaside spheroidal 50-100 nm (thin arrow) elements. They assemble in various degreesin the sectors of the contaminated cells apoptotic or not undergoing progressivedismantling of the peripheral cytoplasm. Fig. 6 - Most of thecytoplasm above the apoptotic nucleus is occupied by membrane bound void spaces ofvarious sizes and forms. The mitochondria are unusually dense, a common occurrencein contaminated cells, apoptotic or not. Adherent to the limiting membrane of thespace indicated (open triangle) a fluffy material possibly derived from MPO.Fig. 7 - Sector of an apoptotic cytoplasm with net-like membranalarrangement as in Fig. 6. Compact mass of parasitic material in a cystic-like form(open triangle). The upper, middle and vertical white lines indicate remnants ofparasitic origin that were not completely removed by the processing of the cells.Part of an elongate membrane bound space possibly still fully occupied by MPO isindicated by the lower line that branches in two.

Mentions: The fine structure of apoptotic HeLa cells from cultures contaminated withmycoplasma in early and in advanced stages of the cell demise process differs from those sofar described in apoptotic cells. The observed changes are enhanced after exposure of thecells to staurosporine. At low microscopic magnifications cells that have apparent normalcytoplasm and nuclei, actually may be harbouring cystic-like profile(s) of parasitic originin an altered cytoplasm. The membranes of the transitional elements of the endoplasmicreticulum (TER) appear fragmented in irregular branching stripes of the smooth component ofthe TER (Fig. 1, white asterisks in L delimitedarea). The concentration of the rough endoplasmic reticulum (RER) membranes is less than innormal HeLa cells. Near to the smooth ER tubule-saccular elements lie groups of 50 nmmicrovesicles aside stacked, thin, various sized profiles of Golgi saccules ( ] ). The 50nm microvesicles bud off mainly from the periphery of the stacked Golgi elements (Fig 1 thin arrow heads inside line U) and also from theextremities of smooth ER tubules (Fig. 1 smallarrows). Small groups of compacted microvesicles are noted in cells still maintainingnormal nuclear appearance (not shown). With the start of chromatin condensationprogressively larger compact microvesicular clusters are formed. These attain sizes larger(Fig. 2) than those of the clusters of microvesicles derived from the fragmentation ofGolgi saccules seen in mitotic (LUCOCQ et al., 1989; SESSO etal., 1999) and in apoptotic (SESSO et al., 1999) cells.Contemporaneously two major cytoplasmic alterations may be noted in contaminated cellsnamely when treated with staurosporine. Occasionally, both deformations appear in the samecell. One, is progressive cytoplasmic loss by formation at the cell periphery of blebs thatseparate from the inner cytoplasm (Figs. 3 and 4) or by localized detachment of sectors ofthe peripheral cytoplasm with various forms and sizes (not shown). In some cells, theremainder thin, cytoplasm with few mitochondria and rough ER profiles surrounds the nucleusin a ring-like form. Such small cells are noted in heavily contaminated samples. Some ofthe cells exhibit sectors of the cytoplasm with a reticulated appearance. Such net-likeregions are composed by various sized tubular and ellipsoidal, apparently empty profiles.It is unclear if the smooth membranes that compose these regions with reticulated aspect,may have derived from the Golgi apparatus. The shape and size of the empty spacescorrespond to those from villus-like formations seen free close to and emerging from thecell surface. In contaminated cells namely after staurosporine treatment the free villus -like forms are seen sprouting from the cell surface and also free nearby. Spheroidal50-100nm (thin arrow in Fig. 5) profiles with inner structure identical to that ofvillus-like elements are consistently proximate to the fake villi. Vestiges of what can beremnants of the villus-simile structures and/or of the parasite itself are seen in thesespaces (Figs. 6 and 7).


Loose and compact agglomerates of 50 nm microvesicles derived from Golgi and endoplasmic reticulum membranes in pre- and in -apoptotic Mycoplasma infected HeLa cells: host-parasite interactions under the transmission electron microscope.

Sesso A, Yamashiro-Kanashiro EH, Orii NM, Taniwaki NN, Kawakami J, Carneiro SM - Rev. Inst. Med. Trop. Sao Paulo (2015 Jan-Feb)

7 are a synopsis of fine structural observations in some 50 samplesof staurosporine (0.1 µM - 2.0 µM for 2 - 24h) and 10 control samples of HeLa cellcultures contaminated with mycoplasma. Fig.1 is from an apparent normal cell at low microscopic exam. Figs 2, 6 and7 and 3 and 4 are from apoptotic cells exposed to staurosporine 0.5 µM/3h and 0.1µM/6h, respectively. Fig. 5 is from a non apoptotic cell exposed to staurosporine0.1 µM/4h. Bars -1 µM. Fig. 1 - HeLa cell-from a heavily contaminated cell culture devoid ofstaurosporine where apoptosis occurred. Normal nucleus (N). Smooth, branched ERprofiles (extremities of thin white lines departing from the central whiteasterisks in the area delimited by the lower (L) dotted line. Conglomerates of 50nm (lower and upper right part of the figure). Part of the microvesicles bud offfrom smooth ER membranes (arrow head) and from tubular elements (small arrows).The majority of the grouped microvesicles pinch off from the extremities ofstacked Golgi saccules (thin arrow heads at the periphery of the frontally exposeddense ellipsoidal-like profile delimited by U)]. Stacked, thin Golgi saccularprofiles of various lengths that often appear dense ( ] ). Fig. 2 -Large, compact conglomeration of various sized microvesicles with predominance ofthe ones with 50 nm. Groups of former dilated stacked Golgi saccules (blackasterisks) are interspersed among the microvesicles. A large such dilated sacculecontains remnants of a parasite or of material of parasitic origin (MPO) (emptytriangle). Fig. 3 - The cytoplasm of this apoptotic cell is delimitedin two concentric major regions. The peripheral one is partitioned into adjacentblebs containing predominantly membranes of the ER. The inner part of thecytoplasm contains clustered, swollen mitochondria. Fig. 4 -Dismantling of the peripheral apoptotic cytoplasm by a contemporaneous detachmentof the previously formed bleb regions. Fig. 5 - Non apoptoticinfected (cysts of parasitic origin, upper arrows) cell, with an abnormallyelongated thin cytoplasm. The free villus-like structures often appear curvedaside spheroidal 50-100 nm (thin arrow) elements. They assemble in various degreesin the sectors of the contaminated cells apoptotic or not undergoing progressivedismantling of the peripheral cytoplasm. Fig. 6 - Most of thecytoplasm above the apoptotic nucleus is occupied by membrane bound void spaces ofvarious sizes and forms. The mitochondria are unusually dense, a common occurrencein contaminated cells, apoptotic or not. Adherent to the limiting membrane of thespace indicated (open triangle) a fluffy material possibly derived from MPO.Fig. 7 - Sector of an apoptotic cytoplasm with net-like membranalarrangement as in Fig. 6. Compact mass of parasitic material in a cystic-like form(open triangle). The upper, middle and vertical white lines indicate remnants ofparasitic origin that were not completely removed by the processing of the cells.Part of an elongate membrane bound space possibly still fully occupied by MPO isindicated by the lower line that branches in two.
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f01: 7 are a synopsis of fine structural observations in some 50 samplesof staurosporine (0.1 µM - 2.0 µM for 2 - 24h) and 10 control samples of HeLa cellcultures contaminated with mycoplasma. Fig.1 is from an apparent normal cell at low microscopic exam. Figs 2, 6 and7 and 3 and 4 are from apoptotic cells exposed to staurosporine 0.5 µM/3h and 0.1µM/6h, respectively. Fig. 5 is from a non apoptotic cell exposed to staurosporine0.1 µM/4h. Bars -1 µM. Fig. 1 - HeLa cell-from a heavily contaminated cell culture devoid ofstaurosporine where apoptosis occurred. Normal nucleus (N). Smooth, branched ERprofiles (extremities of thin white lines departing from the central whiteasterisks in the area delimited by the lower (L) dotted line. Conglomerates of 50nm (lower and upper right part of the figure). Part of the microvesicles bud offfrom smooth ER membranes (arrow head) and from tubular elements (small arrows).The majority of the grouped microvesicles pinch off from the extremities ofstacked Golgi saccules (thin arrow heads at the periphery of the frontally exposeddense ellipsoidal-like profile delimited by U)]. Stacked, thin Golgi saccularprofiles of various lengths that often appear dense ( ] ). Fig. 2 -Large, compact conglomeration of various sized microvesicles with predominance ofthe ones with 50 nm. Groups of former dilated stacked Golgi saccules (blackasterisks) are interspersed among the microvesicles. A large such dilated sacculecontains remnants of a parasite or of material of parasitic origin (MPO) (emptytriangle). Fig. 3 - The cytoplasm of this apoptotic cell is delimitedin two concentric major regions. The peripheral one is partitioned into adjacentblebs containing predominantly membranes of the ER. The inner part of thecytoplasm contains clustered, swollen mitochondria. Fig. 4 -Dismantling of the peripheral apoptotic cytoplasm by a contemporaneous detachmentof the previously formed bleb regions. Fig. 5 - Non apoptoticinfected (cysts of parasitic origin, upper arrows) cell, with an abnormallyelongated thin cytoplasm. The free villus-like structures often appear curvedaside spheroidal 50-100 nm (thin arrow) elements. They assemble in various degreesin the sectors of the contaminated cells apoptotic or not undergoing progressivedismantling of the peripheral cytoplasm. Fig. 6 - Most of thecytoplasm above the apoptotic nucleus is occupied by membrane bound void spaces ofvarious sizes and forms. The mitochondria are unusually dense, a common occurrencein contaminated cells, apoptotic or not. Adherent to the limiting membrane of thespace indicated (open triangle) a fluffy material possibly derived from MPO.Fig. 7 - Sector of an apoptotic cytoplasm with net-like membranalarrangement as in Fig. 6. Compact mass of parasitic material in a cystic-like form(open triangle). The upper, middle and vertical white lines indicate remnants ofparasitic origin that were not completely removed by the processing of the cells.Part of an elongate membrane bound space possibly still fully occupied by MPO isindicated by the lower line that branches in two.
Mentions: The fine structure of apoptotic HeLa cells from cultures contaminated withmycoplasma in early and in advanced stages of the cell demise process differs from those sofar described in apoptotic cells. The observed changes are enhanced after exposure of thecells to staurosporine. At low microscopic magnifications cells that have apparent normalcytoplasm and nuclei, actually may be harbouring cystic-like profile(s) of parasitic originin an altered cytoplasm. The membranes of the transitional elements of the endoplasmicreticulum (TER) appear fragmented in irregular branching stripes of the smooth component ofthe TER (Fig. 1, white asterisks in L delimitedarea). The concentration of the rough endoplasmic reticulum (RER) membranes is less than innormal HeLa cells. Near to the smooth ER tubule-saccular elements lie groups of 50 nmmicrovesicles aside stacked, thin, various sized profiles of Golgi saccules ( ] ). The 50nm microvesicles bud off mainly from the periphery of the stacked Golgi elements (Fig 1 thin arrow heads inside line U) and also from theextremities of smooth ER tubules (Fig. 1 smallarrows). Small groups of compacted microvesicles are noted in cells still maintainingnormal nuclear appearance (not shown). With the start of chromatin condensationprogressively larger compact microvesicular clusters are formed. These attain sizes larger(Fig. 2) than those of the clusters of microvesicles derived from the fragmentation ofGolgi saccules seen in mitotic (LUCOCQ et al., 1989; SESSO etal., 1999) and in apoptotic (SESSO et al., 1999) cells.Contemporaneously two major cytoplasmic alterations may be noted in contaminated cellsnamely when treated with staurosporine. Occasionally, both deformations appear in the samecell. One, is progressive cytoplasmic loss by formation at the cell periphery of blebs thatseparate from the inner cytoplasm (Figs. 3 and 4) or by localized detachment of sectors ofthe peripheral cytoplasm with various forms and sizes (not shown). In some cells, theremainder thin, cytoplasm with few mitochondria and rough ER profiles surrounds the nucleusin a ring-like form. Such small cells are noted in heavily contaminated samples. Some ofthe cells exhibit sectors of the cytoplasm with a reticulated appearance. Such net-likeregions are composed by various sized tubular and ellipsoidal, apparently empty profiles.It is unclear if the smooth membranes that compose these regions with reticulated aspect,may have derived from the Golgi apparatus. The shape and size of the empty spacescorrespond to those from villus-like formations seen free close to and emerging from thecell surface. In contaminated cells namely after staurosporine treatment the free villus -like forms are seen sprouting from the cell surface and also free nearby. Spheroidal50-100nm (thin arrow in Fig. 5) profiles with inner structure identical to that ofvillus-like elements are consistently proximate to the fake villi. Vestiges of what can beremnants of the villus-simile structures and/or of the parasite itself are seen in thesespaces (Figs. 6 and 7).

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Affiliation: Laboratório de Imunopatologia, Instituto de Medicina Tropical (IMT) de São Paulo.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

São Paulo, November 17, 2014 Dear Editor The fine structure of apoptotic HeLa cells from cultures contaminated withmycoplasma in early and in advanced stages of the cell demise process differs from those sofar described in apoptotic cells... These attain sizes larger(Fig. 2) than those of the clusters of microvesicles derived from the fragmentation ofGolgi saccules seen in mitotic (LUCOCQ et al., 1989; SESSO etal., 1999) and in apoptotic (SESSO et al., 1999) cells.Contemporaneously two major cytoplasmic alterations may be noted in contaminated cellsnamely when treated with staurosporine... All cytoplasmic membrane bound organelles as peroxisomes, lysosome-endosomes andthe Golgi apparatus derive from microvesicles that bud off from the ER... The ER is alsomobilized by promoters of cellular stress (references in DOLAI & ADAK, 2014)... The heredescribed structural deviation of the ER-Golgi interface from the normal condition mayrepresent more than only a mycoplasma induced alteration of the programmed cell deathmechanism... It is speculative, whether this initial accumulation of microvesicles in cellswith typical normal nuclei is part of a general forewarning mechanism of cell defence... In aless intense cell stress than that occurring here the observed changes of the TER couldeventually pass undetected under the transmission electron microscope... The mycoplasmas fine structures of our samples are identical (NIR-PAZ etal., 2002; KORNSPAN et al., 2010;) and similar (EDWARDS &FOGH, 1960; HUMMELER et al., 1965; TAYLOR-ROBINSON etal., 1991) to those from various mycoplasmas strains seen in cultures and ininfected cells.

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Related in: MedlinePlus