Golgi clusters and vesicles mediate mitotic inheritance independently of the endoplasmic reticulum

We have examined the fate of Golgi membranes during mitotic inheritance in animal cells using four-dimensional fluorescence microscopy, serial section reconstruction of electron micrographs, and peroxidase cytochemistry to track the fate of a Golgi enzyme fused to horseradish peroxidase. All three approaches show that partitioning of Golgi membranes is mediated by Golgi clusters that persist throughout mitosis, together with shed vesicles that are often found associated with spindle microtubules. We have been unable to find evidence that Golgi membranes fuse during the later phases of mitosis with the endoplasmic reticulum (ER) as a strategy for Golgi partitioning (Zaal, K.J., C.L. Smith, R.S. Polishchuk, N. Altan, N.B. Cole, J. Ellenberg, K. Hirschberg, J.F. Presley, T.H. Roberts, E. Siggia, et al. 1999. Cell. 99:589–601) and suggest that these results, in part, are the consequence of slow or abortive folding of GFP–Golgi chimeras in the ER. Furthermore, we show that accurate partitioning is accomplished early in mitosis, by a process of cytoplasmic redistribution of Golgi fragments and vesicles yielding a balance of Golgi membranes on either side of the metaphase plate before cell division.

Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase.

We have developed a novel technique with which to investigate the morphological basis of exocytotic traffic. We have used expression of HRP from cDNA in a variety of cells in combination with peroxidase cytochemistry to outline traffic into and out of the Golgi apparatus at the electron microscopic level with very high sensitivity. A secretory form of the peroxidase (ssHRP) is active from the beginning of the secretory pathway and the activity is efficiently cleared from cells. Investigation of the morphological elements involved in the itinerary of soluble ER proteins using ssHRP tagged with the ER retention motif (ssHRPKDEL) shows that it progresses through the Golgi stack no further than the cis-most element. Traffic between the RER and the Golgi stack as outlined by ssHRPKDEL occurs via vesicular carriers as well as by tubular elements. ssHRP has also been used to investigate the trans side of the Golgi complex, where incubation at reduced temperatures outlines the trans-Golgi network with HRP reaction product. Tracing the endosomal compartment with transferrin receptor in double-labeling experiments with ssHRP fails to show any overlap between these two compartments.

Abnormal peroxidase-positive granules in “specific granule” deficiency

“Specific granule” deficiency (SGD) has been previously associated with lactoferrin deficiency. The antimicrobial peptides termed defensins, comprising 30% of normal primary granule proteins, have also been shown to be markedly deficient in SGD. The present study was undertaken to correlate these findings with ultrastructural morphometric analysis and peroxidase cytochemistry. Peroxidase-positive, rim-stained, large, defensin-rich dense granules, previously described as a subpopulation of azurophil or primary granules in normal neutrophils, were markedly decreased in a patient with SGD. Morphometric studies of peroxidase- positive granules indicated an average peroxidase-positive granule area (all profiles) in the patient of 0.019 +/- 0.017 micron 2 (mean +/- SD, n = 941) compared to control values from normal neutrophils of two volunteers of 0.049 +/- 0.033 micron 2 (n = 896) and 0.050 +/- 0.039 micron 2 (n = 873) (P less than 0.001 between patient and control samples). Granule histograms showed a single peak of small peroxidase- positive granules, whereas control samples contained more prominent subpopulations of larger peroxidase-positive granules. The total number of peroxidase-positive granules per 100 micron 2 of cytoplasm in the patient was 255 +/- 124 (mean +/- SD, n = 15 cell profiles), which was similar to control values of 266 +/- 63 and 212 +/- 109. Thus, the defensin deficiency in SGD is associated with a decrease in size rather than number of peroxidase-positive granules; suggesting that defensins contribute to normal peroxidase-positive granule size and that SGD is a more global granule deficiency than originally thought.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormal peroxidase-positive granules in “specific granule” deficiency

“Specific granule” deficiency (SGD) has been previously associated with lactoferrin deficiency. The antimicrobial peptides termed defensins, comprising 30% of normal primary granule proteins, have also been shown to be markedly deficient in SGD. The present study was undertaken to correlate these findings with ultrastructural morphometric analysis and peroxidase cytochemistry. Peroxidase-positive, rim-stained, large, defensin-rich dense granules, previously described as a subpopulation of azurophil or primary granules in normal neutrophils, were markedly decreased in a patient with SGD. Morphometric studies of peroxidase- positive granules indicated an average peroxidase-positive granule area (all profiles) in the patient of 0.019 +/- 0.017 micron 2 (mean +/- SD, n = 941) compared to control values from normal neutrophils of two volunteers of 0.049 +/- 0.033 micron 2 (n = 896) and 0.050 +/- 0.039 micron 2 (n = 873) (P less than 0.001 between patient and control samples). Granule histograms showed a single peak of small peroxidase- positive granules, whereas control samples contained more prominent subpopulations of larger peroxidase-positive granules. The total number of peroxidase-positive granules per 100 micron 2 of cytoplasm in the patient was 255 +/- 124 (mean +/- SD, n = 15 cell profiles), which was similar to control values of 266 +/- 63 and 212 +/- 109. Thus, the defensin deficiency in SGD is associated with a decrease in size rather than number of peroxidase-positive granules; suggesting that defensins contribute to normal peroxidase-positive granule size and that SGD is a more global granule deficiency than originally thought.(ABSTRACT TRUNCATED AT 250 WORDS)

The development of the resident pattern of endogenous peroxidatic activity in mouse peritoneal macrophages coincides with the expression of the differentiation antigen F4/80. A combined method for immunoperoxidase labeling and endogenous peroxidase cytochemistry.

In the mouse the maturation of mononuclear phagocytes was followed by comparing the ultrastructural pattern of endogenous peroxidatic activity (PA) at different time points during an acute peritonitis induced with newborn calf serum (NCS). Exudate macrophages demonstrate PA only in lysosomes, whereas resident macrophages have reaction product in the nuclear envelope (NE) and rough endoplasmic reticulum (RER). Transitional cells called "exudate-resident" macrophages have PA in the NE, RER, and some virginal lysosomes. In addition, peroxidase-negative macrophages were also present. A monoclonal antibody, F4/80, that specifically recognizes a mouse macrophage differentiation antigen (Austyn and Gordon, 1981) was used in this study. To compare the indirect immunoperoxidase labeling of this antigen and the endogenous peroxidase cytochemistry on the cellular level, a combined method was developed. Finally, the method was applied to the peritoneal cells at different time points after intraperitoneal injection of NCS in mice. The relative numbers of cells demonstrating the different patterns of endogenous PA and the proportions of each subpopulation expressing F4/80 antigen were estimated. It appeared that the expression of the antigen F4/80 coincides with the development of the resident pattern of PA. It is therefore concluded that the macrophages with the resident pattern of endogenous peroxidase are derived from monocyte-like exudate macrophages. In addition, the results indicate that both exudate-resident macrophages and at least a part of the peroxidase-negative macrophages are transitional forms.

Transcellular transport of polymeric IgA in the rat hepatocyte: biochemical and morphological characterization of the transport pathway.

Polymeric IgA (pIgA) is transported by liver parenchymal cells (hepatocytes) from blood to bile via a receptor-mediated process. We have studied the intracellular pathway taken by a TEPC15 mouse myeloma pIgA. When from 1 microgram to 1 mg 125I-pIgA was injected into the saphenous vein of a rat, 36% was transported as intact protein into the bile over a 3-h period. The concentration of transported 125I-pIgA was maximal in bile 30-60 min after injection, and approximately 80% of the total 125I-pIgA ultimately transported had been secreted into bile by 90 min. A horseradish peroxidase-pIgA conjugate (125I-pIgA-HRP) was transported to a similar extent and with kinetics similar to that of unconjugated 125I-pIgA and was therefore used to visualize the transport pathway. Peroxidase cytochemistry of livers fixed in situ 2.5 to 10 min after 125I-pIgA-HRP injection demonstrated a progressive redistribution of labeled structures from the sinusoidal area to intermediate and bile canalicular regions of the hepatocyte cytoplasm. Although conjugate-containing structures began accumulating in the bile canalicular region at these early times, no conjugate was present in bile until 20 min. From 7.5 to 45 min after injection approximately 30% of the labeled structures were in regions that contained Golgi complexes and lysosomes; however, we found no evidence that either organelle contained 125I-pIgA-HRP. At least 85% of all positive structures in the hepatocyte were vesicles of 110-160-nm median diameters, with the remaining structures accounted for by tubules and multivesicular bodies. Vesicles in the bile canalicular region tended to be larger than those in the sinusoidal region. Serial sectioning showed that the 125I-pIgA-HRP-containing structures were relatively simple (predominantly vesicular) and that extensive interconnections did not exist between structures in the sinusoidal and bile canalicular regions.