title: Golgi proteins in circulating human platelets are distributed across non-stacked, scattered structures.
abstract: Platelets are small, anucleate cell fragments that are central to hemostasis, thrombosis, and inflammation. They are derived from megakaryocytes from which they inherit their organelles. As platelets can synthesize proteins and contain many of the enzymes of the secretory pathway, one might expect all mature human platelets to contain a stacked Golgi apparatus, the central organelle of the secretory pathway. By thin section electron microscopy, stacked membranes resembling the stacked Golgi compartment in megakaryocytes and other nucleated cells can be detected in both proplatelets and platelets. However, the incidence of such structures is low and whether each and every platelet contains such a structure remains an open question. By single-label, immunofluorescence staining, Golgi glycosyltransferases are found within each platelet and map to scattered structures. Whether these structures are positive for marker proteins from multiple Golgi subcompartments remains unknown. Here, we have applied state-of-the-art techniques to probe the organization state of the Golgi apparatus in resting human platelets. By the whole cell volume technique of serial-block-face scanning electron microscopy (SBF-SEM), we failed to observe stacked, Golgi-like structures in any of the 65 platelets scored. When antibodies directed against Golgi proteins were tested against HeLa cells, labeling was restricted to an elongated juxtanuclear ribbon characteristic of a stacked Golgi apparatus. By multi-label immunofluorescence microscopy, we found that each and every resting human platelet was positive for cis, trans, and trans Golgi network (TGN) proteins. However, in each case, the proteins were found in small puncta scattered about the platelet. At the resolution of deconvolved, widefield fluorescence microscopy, these proteins had limited tendency to map adjacent to one another. When the results of 3D structured illumination microscopy (3D SIM), a super resolution technique, were scored quantitatively, the Golgi marker proteins failed to map together indicating at the protein level considerable degeneration of the platelet Golgi apparatus relative to the layered stack as seen in the megakaryocyte. In conclusion, we suggest that these results have important implications for organelle structure/function relationships in the mature platelet and the extent to which Golgi apparatus organization is maintained in platelets. Our results suggest that Golgi proteins in circulating platelets are present within a series of scattered, separated structures. As separate elements, selective sets of Golgi enzymes or sugar nucleotides could be secreted during platelet activation. The establishment of the functional importance, if any, of these scattered structures in sequential protein modification in circulating platelets will require further research.
authors.affiliation: a Department of Physiology and Biophysics , University of Arkansas for Medical Sciences , Little Rock , AR , USA.
authors.forename: Shilpi
authors.initials: S
authors.lastname: Yadav
authors.affiliation: a Department of Physiology and Biophysics , University of Arkansas for Medical Sciences , Little Rock , AR , USA.
authors.forename: Jonathan K
authors.initials: JK
authors.lastname: Williamson
authors.affiliation: b National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health , Bethesda , MD , USA.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Aronova
authors.affiliation: a Department of Physiology and Biophysics , University of Arkansas for Medical Sciences , Little Rock , AR , USA.
authors.forename: Andrew A
authors.initials: AA
authors.lastname: Prince
authors.affiliation: a Department of Physiology and Biophysics , University of Arkansas for Medical Sciences , Little Rock , AR , USA.
authors.forename: Irina D
authors.initials: ID
authors.lastname: Pokrovskaya
authors.affiliation: b National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health , Bethesda , MD , USA.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Leapman
authors.affiliation: a Department of Physiology and Biophysics , University of Arkansas for Medical Sciences , Little Rock , AR , USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Storrie
mesh_terms: D001792:Blood Platelets / Q000378:metabolism*; D018929:Cell Culture Techniques; D006056:Golgi Apparatus / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015388:Organelles
keywords: 3D SIM; Golgi apparatus; fluorescence microscopy; platelets; serial-block-face scanning electron microscopy