Histochemistry and Cell Biology / Golgi apparatus dis- and reorganizations studied with the aid of 2-deoxy-d-glucose and visualized by 3D-electron tomography

We studied Golgi apparatus disorganizations and reorganizations in human HepG2 hepatoblastoma cells by using the nonmetabolizable glucose analogue 2-deoxy-d-glucose (2DG) and analyzing the changes in Golgi stack architectures by 3D-electron tomography. Golgi stacks remodel in response to 2DG-treatment and are replaced by tubulo-glomerular Golgi bodies, from which mini-Golgi stacks emerge again after removal of 2DG. The Golgi stack changes correlate with the measured ATP-values. Our findings indicate that the classic Golgi stack architecture is impeded, while cells are under the influence of 2DG at constantly low ATP-levels, but the Golgi apparatus is maintained in forms of the Golgi bodies and Golgi stacks can be rebuilt as soon as 2DG is removed. The 3D-electron microscopic results highlight connecting regions that interlink membrane compartments in all phases of Golgi stack reorganizations and show that the compact Golgi bodies mainly consist of continuous intertwined tubules. Connections and continuities point to possible new transport pathways that could substitute for other modes of traffic. The changing architectures visualized in this work reflect Golgi stack dynamics that may be essential for basic cell physiologic and pathologic processes and help to learn, how cells respond to conditions of stress.(VLID)353187

Human endothelial progenitor cells internalize high-density lipoprotein.

Endothelial progenitor cells (EPCs) originate either directly from hematopoietic stem cells or from a subpopulation of monocytes. Controversial views about intracellular lipid traffic prompted us to analyze the uptake of human high density lipoprotein (HDL), and HDL-cholesterol in human monocytic EPCs. Fluorescence and electron microscopy were used to investigate distribution and intracellular trafficking of HDL and its associated cholesterol using fluorescent surrogates (bodipy-cholesterol and bodipy-cholesteryl oleate), cytochemical labels and fluorochromes including horseradish peroxidase and Alexa Fluor® 568. Uptake and intracellular transport of HDL were demonstrated after internalization periods from 0.5 to 4 hours. In case of HDL-Alexa Fluor® 568, bodipy-cholesterol and bodipy-cholesteryl oleate, a photooxidation method was carried out. HDL-specific reaction products were present in invaginations of the plasma membrane at each time of treatment within endocytic vesicles, in multivesicular bodies and at longer periods of uptake, also in lysosomes. Some HDL-positive endosomes were arranged in form of "strings of pearl"- like structures. HDL-positive multivesicular bodies exhibited intensive staining of limiting and vesicular membranes. Multivesicular bodies of HDL-Alexa Fluor® 568-treated EPCs showed multilamellar intra-vacuolar membranes. At all periods of treatment, labeled endocytic vesicles and organelles were apparent close to the cell surface and in perinuclear areas around the Golgi apparatus. No HDL-related particles could be demonstrated close to its cisterns. Electron tomographic reconstructions showed an accumulation of HDL-containing endosomes close to the trans-Golgi-network. HDL-derived bodipy-cholesterol was localized in endosomal vesicles, multivesicular bodies, lysosomes and in many of the stacked Golgi cisternae and the trans-Golgi-network Internalized HDL-derived bodipy-cholesteryl oleate was channeled into the lysosomal intraellular pathway and accumulated prominently in all parts of the Golgi apparatus and in lipid droplets. Subsequently, also the RER and mitochondria were involved. These studies demonstrated the different intracellular pathway of HDL-derived bodipy-cholesterol and HDL-derived bodipy-cholesteryl oleate by EPCs, with concomitant

Micrographs of EPCs incubated with HDL labeld bodipy-cholesterol between 30 and 240 min.

<p><b>A</b>) Small cytoplasmic vesicles are stained (black thin arrows), the large 2 positive MVBs exhibit tightly-packed intraluminal microvesicles with reaction products. <b>B</b>), <b>C</b>) Reaction products are widely dispersed in all parts of the Golgi apparatus and the TGN. <b>D</b>) RER (white arrows) and mitochondria (M and in inset) are weakly stained. <b>E</b>) Demonstration of close association between unlabeled lipid droplets and RER. (Scale bar: A = 0.5 µm; B = 1 µm; C, D, D inset, E = 0.25 µm).</p

Ultrastructural appearance of EPCs after 30 minute of HDL-HRP internalization.

<p><b>A</b>) HDL-HRP particles can be seen at the cell surface (black arrow), the various sizes of HDL-HRP positive endosomal vesicles (E) are present beneath the cell membrane and close to the Golgi apparatus (G). <b>B</b>) HDL-positive and -negative MVBs (MVBs, asterisk) are visible. The white arrowhead indicates HDL-negative MVBs representing an early stage of lipid internalization; lipid droplets (Li) appear unstained. <b>C</b>) Lysosomes (L) shows intraluminal HDL-HRP particles. <b>D</b>) The RER is HDL-HRP negative while two invaginations of the plasma membrane (black arrowheads) show a strong reaction. (Scale bar: A, C = 1 µm; B, D = 0.5 µm).</p

Time course of HDL-Alexa Fluor® 568 internalization after interval from 30 to 240 min shows the fluorescence-marked structures in bright signals increasing in size and number of intracellular endocytic compartments.

<p>(Scale bar: A, B, C, D = 50 µm)</p

ET indicates HDL-containing endosomes.

<p><b>A</b>) shows a virtual slice, <b>B</b>) a corresponding model with endosomes (bright blue color) are accumulated in close proximity to the Golgi apparatus (orange color). (Scale bar: A, B = 200 µm).</p

Micrographs of EPCs at 1 hour of incubation with HDL-HRP.

<p><b>A</b>) Numerous positive endosomal vesicles of varying sizes are found throughout cytoplasm, membrane invagination are indicated by arrowheads, positive MVBs are present. <b>B</b>) Positive MVBs with irregular shape and tubular appendices (white thin arrows), an endosome alignment (black thin arrows) as well as membrane invagination (black arrowhead) are demonstrated. <b>C</b>) Showing positively and negatively stained MVBs (asterisk). <b>D</b>) The large structures represent autophagosomes (white thick arrows) containing endosomes, are enclosed by a multilamellar membrane. <b>E</b>) The HDL-HRP particles are apparent in lysosomes (L, black thick arrow) (Scale bar: A, B, E = 1 µm; C, D = 0.5 µm).</p

Ultrastructural appearance of EPCs after 4 hour of incubation with HDL-HRP.

<p><b>A</b>) Autophagosomes (white thick arrows) with engulfed endosomal vesicles are noted. <b>B</b>) Positive MVBs with the characteristic tubular membranous extensions are visible. <b>C</b>) High magnification of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083189#pone-0083189-g006" target="_blank">figure 6a</a>, shows a large spherical secondary lysosomes (L) close to Golgi apparatus (G). <b>D</b>) Demonstration of “strings of pearls-like structures”. (Scale bar: A = 1 µm; B, C, D = 0.5 µm).</p