N-聚糖和O-聚糖在极性化细胞蛋白质分拣中的作用

极性化上皮细胞的质膜因其所含蛋白质、脂质等组分不同,可以分为细胞膜顶端和细胞膜基底侧端两个区域,而新合成的蛋白质向这两个区域的有效分拣是上皮细胞维持其自身极性及正常功能所必需的。细胞膜基底侧端蛋白质的分拣主要由位于该蛋白质胞质区的信号肽所介导,关于这方面的研究是比较深入的;而细胞膜顶端蛋白质的分拣机制目前尚未阐明,因而显得比较复杂。近年来,糖类分子作为生物体内细胞识别和调控过程的信息分子日益受到关注,人们通过干扰聚糖合成、基因突变以及构建糖基化缺陷细胞株等实验方法,逐渐地认识到糖类分子在极性化上皮细胞的蛋白质分拣调节中起重要作用。由于糖分子本身结构非常复杂,而且目前缺乏研究糖类分子的有效手段,使得糖生物学的研究远远落后于蛋白质和核酸的研究。从而导致探讨糖类分子在蛋白质分拣过程的具体机制相对来说比较困难。本综述拟简要概括糖类分子中N-聚糖和O-聚糖在极性化上皮细胞的蛋白质分拣过程中的作用,以及两种聚糖在此过程中行使分拣信号功能的可能机制。     

Analysis of bivariate flow karyotypes

Bivariate flow karyotype analysis is performed using data from chromosomes stained with two fluorescent dyes, typically chromomycin A3 and Hoechst-33258, and measured in a flow cytometer or cell sorter (Carrano et al.: Proceedings of the National Academy of Sciences of the United States of America 76:1382-1384, 1979; Gray et al.: Proceedings of the National Academy of Sciences of the United States of America 72:1231-1234, 1975; Langlois et al.: Proceedings of the National Academy of Sciences of the United States of America 79:7876-7880, 1982). In the resulting bivariate histogram, most chromosome types appear as individual peaks. In sorting of chromosomes to purify a specific chromosomal type, its corresponding peak in the bivariate histogram is delineated by a rectangular region which surrounds it. All events (objects) that fall within this region trigger the sorting process. In most cases, peaks for different chromosomal types overlap to some extent, and in addition there is always an underlying background due to chromosome fragments and clumps. Thus the sorted population will not be pure; it may include more than one chromosome type and will include debris. To determine the purity of a sort, i.e., the percentage of the sorted material that is of the actual chromosomal type desired, two methods of mathematical analysis have been developed. In the more general method, the bivariate data within an analysis region that includes the sort region, are fit with a series of bivariate Gaussian functions, one for each peak. In a simplified method, the data within the analysis region are transformed into a univariate distribution of either chromomycin A3 or Hoechst-33258 fluorescence. The peaks in these univariate distributions are fit with univariate Gaussian functions. In both methods the purity is determined mathematically. The results of both methods agree well with independent methods of analysis.     

Rapid assay for cell age response to radiation by electronic volume flow cell sorting

A new technique is described for measuring cell survival as a function of cell cycle position using flow cytometric cell sorting on the basis of electronic volume signals. The sorting of cells into different cell age compartments is demonstrated for three different cell lines commonly used in radiobiological research. Using flow cytometric DNA content analysis and [3H]thymidine autoradiography of the sorted cell populations, we demonstrate that the resolution of the age compartment separation is as good as or better than that reported for other cell synchronizing techniques. The variation in cell survival as a function of position in the cell cycle after a single dose of radiation as measured by volume cell sorting is similar to that determined by other cell synchrony techniques. This new method has several advantages, including: no treatment of the cells is required, thus, this method is noncytotoxic; no cell cycle progression is needed to obtain different cell age compartments; the cell population can be held in complete growth medium at any desired temperature during sorting; and a complete radiation age-response assay can be plated in 2 h. The application of this method to problems in radiobiology and chemotherapy is discussed, along with some of the technical limitations.     

Cell sorting within the prestalk zone of Dictyostelium discoideum

Abstract. The prestalk zone of slugs of Dictyostelium discoideum has been shown to contain three subregions in which the extracellular matrix genes ecmA and ecmB are differentially expressed; it is generally thought that these regions are defined by extracellular signals. Using β-galactosidase as a cell marker, we have shown that cells can sort specifically to all three regions. Cells from the posterior-prestalk zone ("prestalk 0 zone") which are injected into the slug tip move within 60 min back to their position of origin. When cells from the anterior prestalk zone (presumably containing a mixture of ecmA and ecmB expressers) are transplanted to the posterior prestalk zone, they move to the tip ("prestalk A zone") within 1 h and about 30 min subsequently are often found in a cone-shaped region within the tip ("prestalk B zone"). Cells transplanted to their own positions do not move significantly within this period. Since the sub-regions of the prestalk zone can be defined by sorting, it is possible that they are normally formed in this way rather than by position-dependent signals. Cells transplanted without a change in anterior-posterior position and cells which have sorted back to their positions of origin eventually spread out throughout the prestalk zone. This suggests that sorting preferences of cells are respecified. When posterior prestalk cells are transplanted to the prespore zone, respecification of sorting preference is suspended until the cells return to the prestalk zone and anterior-prestalk cells acquire posterior-prestalk sorting preferences.     

Chromosomes for molecular hybridization. Assignment of repetitive and single copy genes using a rapid filter-fixation method

Specific recombinant DNA sequences (5S rRNA, B1, albumin) were assigned to flow sorted chromosomes of the Chinese hamster cell line CHV79. For this purpose, a rapid protocol was developed using filterbound chromosomal DNA and probing with various nucleic acids, that allows sequence identification in chromosomes. A flow histogram and a flow karyogram of the CHV79 cell line were established by flow analysis in order to calculate the amount of DNA per CHV79 cell and their chromosomes. Subsequently, metaphase chromosomes or chromosomal groups were fractionated by electronic sorting and a defined number of chromosomes was directly bound to nitrocellulose filters for sequence homology analysis by a dot blot hybridization procedure. This procedure not only allows the assigning of specific DNA sequences to particular chromosomes, it is also applicable to studies of changes in karyotypes, for example translocations of given sequences.     

Flow sorting from organ material by intracellular markers.

BACKGROUND: Fluorescence-activated cell sorting (FACS) is an attractive technique for gene or protein expression studies in rare cell populations. For cell types where specific surface markers are not known, intracellular markers can be used. However, this approach is currently held to be difficult, as the required fixation and permeabilization may cause protein modification and RNA degradation. METHODS AND RESULTS: Using the rat thyroid gland as model, rare (parafollicular) and frequent (follicular) endocrine cell types were sorted based on immunostaining for intracellular calcitonin peptide and thyroglobulin protein expression. The sorted cells were compatible with Western blot analysis of proteins, immunoassay detection of calcitonin peptide hormone and RT-PCR. CONCLUSION: We developed a robust FACS protocol that allows flow sorting of rare cells from dissociated organ material, based on intracellular markers. Our FACS protocol is compatible with downstream analysis of proteins, peptides, and mRNA in the sorted cells.     

Developmental age-related cell sorting inDictyostelium discoideum

Summary The sorting behavior of mixtures ofD. discoideum cells which had been developed for different lengths of time was examined. Cells developed for 4 and 8 h were mixed together and allowed to form slugs. Within the slugs, 8 h cells sorted to the anterior prestalk region while 4 h cells sorted to the posterior prespore region. These results indicate that the more developed a cell is, the more likely it is to become part of the prestalk zone in the slug. They are also consistent with the differential adhesion and chemotaxis hypotheses as a mechanism for cell sorting since cells become more adhesive and chemotactically responsive as development proceeds.     

A cytoplasmic sequence in human tyrosinase defines a second class of di-leucine-based sorting signals for late endosomal and lysosomal delivery

Distinct cytoplasmic sorting signals target integral membrane proteins to late endosomal compartments, but it is not known whether different signals direct targeting by different pathways. The availability of multiple pathways may permit some cell types to divert proteins to specialized compartments, such as the melanosome of pigmented cells. To address this issue, we characterized sorting determinants of tyrosinase, a tissue-specific resident protein of the melanosome. The cytoplasmic domain of tyrosinase was both necessary and sufficient for internalization and steady state localization to late endosomes and lysosomes in HeLa cells. Mutagenesis of two leucine residues within a conventional di-leucine motif ablated late endosomal localization. However, the properties of this di-leucine-based signal were distinguished from that of CD3gamma by overexpression studies; overexpression of the tyrosinase signal, but not the well characterized CD3gamma signal, induced a 4-fold enlargement of late endosomes and lysosomes and interfered with endosomal sorting mediated by both tyrosine- and other di-leucine-based signals. These properties suggest that the tyrosinase and CD3gamma di-leucine signals are distinctly recognized and sorted by distinct pathways to late endosomes in non-pigmented cells. We speculate that melanocytic cells utilize the second pathway to divert proteins to the melanosome.     

Short-term physiologic effects of mechanical flow sorting and the Becton-Dickinson cell concentrator in cultures of the marine phytoflagellata Emiliania huxleyi and Micromonas pusilla.

BACKGROUND: In contrast to large, high-efficiency cytometers, mechanically sorting benchtop instruments provide a feasible alternative for shipboard cell sorting of oceanic microbial communities. However, sorting efficiency of these instruments is constrained by their maximum sorting rate of approximately 300 cells/s and by constant dilution of sorted samples by sheath flow. These factors often render too low sorted cell concentrations for postsorting experiments of oceanic phytoplankton populations of low natural abundance. A Cell Concentrator module has been marketed to overcome these dilution effects. Postsorting experiments also have to consider potential physiologic effects of cell sorting. Short-term physiologic effects on phytoplankton photosynthetic rates and esterase activities by mechanical flow sorting and cell concentration and on the efficiency of the Cell Concentrator module are evaluated. METHODS: Increasing numbers of the oceanic phytoflagellates Micromonas pusilla and Emiliania huxleyi were sorted and concentrated, and recovery in the concentrated samples was compared with the sorted-only samples (concentration rate) and the total number of sorted cells (recovery rate). Photosynthetic rates and metabolic activities of sorted and sorted/concentrated cells were compared with unsorted cells. Photosynthetic rates were estimated from 14CO2 uptake experiments and metabolic activity quantified cytometrically after cleavage of fluorescein diacetate. RESULTS: Irrespective of the total number of sorted cells, concentration rates between concentrated and sorted cells remained mostly below 10-fold and did not increase with the number of concentrated cells. Recovery rates in the concentrated samples amounted to fewer than 10% of total sorted cells, except for forceful resuspension attempts in the Concentrator insert (25-44%), which might be unsuitable for delicate species. Cell sorting resulted in a 24-49% decrease in photosynthetic rates. Metabolic activity within metabolically active cells was not affected by cell sorting, but the share of metabolically active cells decreased by 32-37%. Cell concentration did not affect metabolic activity or the fraction of active cells but did increase photosynthetic rate several-fold compared with unsorted cells. CONCLUSION: Low recovery of concentrated cells, probably due to cell adhesion to the filer bottom of the Concentrator insert, render the Cell Concentrator of limited use to overcome dilution problems of mechanical flow sorting, particularly when results are extrapolated to natural, low-abundance populations. Severe changes in photosynthetic rates also render concentrated cells suspicious for subsequent physiologic experiments. Mechanical sorting alone also exhibited significant physiologic effects on sorted cells, some of which might not be temporary. Comparable effects between mechanical sorting and droplet sorting as previously reported confirm that physiologic effects might be caused predominantly by shear stress and laser exposure during cytometric analysis rather than the sorting process. Sufficient recovery time must be allowed before postsorting experiments, but potential changes in cell physiology from the natural conditions during postsorting recovery must be considered.     

Automatic cell identification and enrichment in lung cancer: V. Adenocarcinoma and large cell undifferentiated carcinoma

The aims of this study were to develop a protocol for the identification and enrichment of cancer cells from sputum obtained from patients with adenocarcinoma of the lung (n = 6) and large-cell undifferentiated carcinoma of the lung (n = 2), and to compare these findings with the results from our previous studies on other cell types from lung cancer. The hypotheses tested were: Cancer cells in sputum can be preserved following flow sorting. Enrichment for cancer cells from acridine orange (AO)-stained specimens can be achieved. Discrimination of cancer cells from noncancer cells is by AO green fluorescence and discrimination of lymphocytes from other cell types is by AO red fluorescence. Cancer cells are consistently enriched in the AO high green and red fluorescence region, although, for a given cell type, maximal enrichment is patient-dependent. Finally, cancer cell enrichment and lymphocyte exclusion can be done simultaneously. Cells from sputum were initially fixed, stained with AO, sorted on a dual parameter flow sorter, and classified into six groups corresponding to two ranges of green and three ranges of red fluorescence intensities. Cells of each region were stained by the method of Papanicolaou and differential counts were performed to determine the relative frequencies (i.e., purities) of leukocytes, macrophages, squamous cells, and cancer cells, in sorted and unsorted (i.e., control) samples. The average purity of leukocytes (81%), macrophages (6%), squamous cells (11%), and cancer cells (2%) varied markedly from sample to sample. However, the largest enrichment values (i.e., ratio of purity of a cell type in a sorted sample to its purity in the unsorted control sample) achieved for cancer cells consistently occurred for each patient sample in the region corresponding to high green and high red fluorescence intensities. Experimentally, a cancer cell average enrichment of sixteen-fold was obtained by this method. Additionally, fluorescence intensity ranges which increased the enrichment for macrophages by cell sorting typically excluded leukocytes and squamous cells, and vice versa. Finally, red fluorescence intensity was the primary discriminatory parameter for all cell types studied, although the additional use of green fluorescence intensity significantly increased cancer cell enrichment rates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation and Characterization of a Primary Proximal Tubular Epithelial Cell Model from Human Kidney by CD10/CD13 Double Labeling

Renal proximal tubular epithelial cells play a central role in renal physiology and are among the cell types most sensitive to ischemia and xenobiotic nephrotoxicity. In order to investigate the molecular and cellular mechanisms underlying the pathophysiology of kidney injuries, a stable and well-characterized primary culture model of proximal tubular cells is required. An existing model of proximal tubular cells is hampered by the cellular heterogeneity of kidney; a method based on cell sorting for specific markers must therefore be developed. In this study, we present a primary culture model based on the mechanical and enzymatic dissociation of healthy tissue obtained from nephrectomy specimens. Renal epithelial cells were sorted using co-labeling for CD10 and CD13, two renal proximal tubular epithelial markers, by flow cytometry. Their purity, phenotypic stability and functional properties were evaluated over several passages. Our results demonstrate that CD10/CD13 double-positive cells constitute a pure, functional and stable proximal tubular epithelial cell population that displays proximal tubule markers and epithelial characteristics over the long term, whereas cells positive for either CD10 or CD13 alone appear to be heterogeneous. In conclusion, this study describes a method for establishing a robust renal proximal tubular epithelial cell model suitable for further experimentation.     

The cell adhesion molecule DdCAD-1 regulates morphogenesis through differential spatiotemporal expression in Dictyostelium discoideum

During development of Dictyostelium, multiple cell types are formed and undergo a coordinated series of morphogenetic movements guided by their adhesive properties and other cellular factors. DdCAD-1 is a unique homophilic cell adhesion molecule encoded by the cadA gene. It is synthesized in the cytoplasm and transported to the plasma membrane by contractile vacuoles. In chimeras developed on soil plates, DdCAD-1-expressing cells showed greater propensity to develop into spores than did cadA-null cells. When development was performed on non-nutrient agar, wild-type cells sorted from the cadA-null cells and moved to the anterior zone. They differentiated mostly into stalk cells and eventually died, whereas the cadA-null cells survived as spores. To assess the role of DdCAD-1 in this novel behavior of wild-type and mutant cells, cadA-null cells were rescued by the ectopic expression of DdCAD-1-GFP. Morphological studies have revealed major spatiotemporal changes in the subcellular distribution of DdCAD-1 during development. Whereas DdCAD-1 became internalized in most cells in the post-aggregation stages, it was prominent in the contact regions of anterior cells. Cell sorting was also restored in cadA(-) slugs by exogenous recombinant DdCAD-1. Remarkably, DdCAD-1 remained on the surface of anterior cells, whereas it was internalized in the posterior cells. Additionally, DdCAD-1-expressing cells migrated slower than cadA(-) cells and sorted to the anterior region of chimeric slugs. These results show that DdCAD-1 influences the sorting behavior of cells in slugs by its differential distribution on the prestalk and prespore cells.     

Mechanisms of apical protein sorting in polarized thyroid epithelial cells.

The process leading to thyroid hormone synthesis is vectorial and depends upon the polarized organization of the thyrocytes into the follicular unit. Thyrocyte membrane proteins are delivered to two distinct domains of the plasma membrane using apical (AP) and basolateral (BL) sorting signals. A recent hypothesis for AP sorting proposes that apically destined proteins cluster with glycosphingolipids (GSLs) and cholesterol, into microdomains (or rafts) of the Golgi membrane from which AP vesicles originate. In MDCK cells the human neurotrophin receptor, p75hNTR, is delivered to the AP surface through a sorting signal, rich in O-glycosylated sugars, identified in its ectodomain. We have investigated whether this signal is functional in the thyroid-derived FRT cell line and whether p75hNTR clusters into lipid rafts to be sorted to the AP membrane. We found that p75hNTR is apically delivered via a direct pathway and does not associate with rafts during its transport to the surface of FRT cells. Therefore, although the same signal could be recognized by different cell types thyroid cells may possess a tissue-specific sorting machinery.     

Lysosome rich cells contain the lytic activity of lymphokine-activated killer cell populations

Summary Little is known regarding the effectors of lymphokine-activated killer activity. Lysosomotropic agents such as quinacrine can be used to positively sort for lysosome rich cells in natural killer (NK) cell populations. We therefore decided to use this agent to sort lymphokine-activated killer (LAK) cells to characterize their lysosomal content. We found that the positively sorted population contained all the LAK activity, i.e., lysis of NK-resistant tumor cells (B16 melanoma cell line), with the negatively sorted cells having no killing activity. Therefore separation of interleukin-2-incubated cells for LAK activity could be accomplished using sorting after quinacrine staining. The treatment of positively sorted LAK cell populations with L-leucine methyl ester, a lysosomotropic dye which inhibits killing by lysosome rich cells, caused abrogation of killing of the B16 tumor by the treated populations. Single cell conjugate assays were also done on these sorted cells, with positively sorted cells forming the highest and negatively sorted cells the lowest percent of conjugates. Our data therefore indicates the important role of lysosome rich cells in the LAK cell population in the murine system.This work was supported by NIH grants R01 CA42962 and K04 CA0122, and by intramural funds from the Norris Cancer Center     

Purification of the chromosomes of the Indian muntjac by flow sorting.

Metaphase chromosomes were isolated from a male Indian muntjac cell line, were stained with ethidium bromide and were analyzed by flow microfluorometry to establish a deoxyribonucleic acid (DNA)-based karyotype. Five major peaks were evident on the chromosomal DNA distribution corresponding to the five chromosome types in this species. The amount of DNA in each chromosome was confirmed by cytophotometric measurements of intact metaphase spreads. The five chromosome types were separated by flow sorting at rates up to several hundred chromosomes per second. The sorted chromosomes were identified by morphology and by Giemsa banding patterns. The automsomes, Numbers 1, 2 and 3, and the X + 3 composite chromosome were separated with a high degree of purity (90%). The centromere region of the X + 3 chromosome was fragile to mechanical shearing, and during isolation a small proportion of these chromosomes broke into four segiments: the long arm, the short arm, the short arm plus centromere and the centromere region. A large fraction of the constitutive heterochromatin of this species is present in the centromere region of the X + 3 chromosome and in the Y chromosome; these two regions possess similar amounts of DNA and therefore sort together. Chromosome flow sorting is rapid, reproducible and precise; it allows the collection of microgram quantities of purified chromosomes.     

Chromosomes for molecular hybridization

Summary Specific recombinant DNA sequences (5S rRNA, B1, albumin) were assigned to flow sorted chromosomes of the Chinese hamster cell line CHV79. For this purpose, a rapid protocol was developed using filterbound chromosomal DNA and probing with various nucleic acids, that allows sequence identification in chromosomes. A flow histogram and a flow karyogram of the CHV79 cell line were established by flow analysis in order to calculate the amount of DNA per CHV79 cell and their chromosomes. Subsequently, metaphase chromosomes or chromosomal groups were fractionated by electronic sorting and a defined number of chromosomes was directly bound to nitrocellulose filters for sequence homology analysis by a dot blot hybridization procedure. This procedure not only allows the assigning of specific DNA sequences to particular chromosomes, it is also applicable to studies of changes in karyotypes, for example translocations of given sequences.Some results shown constituted a Diploma Thesis by G.L. submitted to and accepted by the Department of Biology, University of Kaiserslautern     

Sorting things out: Assessing effects of unequal specimen biomass on DNA metabarcoding

Environmental bulk samples often contain many different taxa that vary several orders of magnitude in biomass. This can be problematic in DNA metabarcoding and metagenomic high‐throughput sequencing approaches, as large specimens contribute disproportionately high amounts of DNA template. Thus, a few specimens of high biomass will dominate the dataset, potentially leading to smaller specimens remaining undetected. Sorting of samples by specimen size (as a proxy for biomass) and balancing the amounts of tissue used per size fraction should improve detection rates, but this approach has not been systematically tested. Here, we explored the effects of size sorting on taxa detection using two freshwater macroinvertebrate bulk samples, collected from a low‐mountain stream in Germany. Specimens were morphologically identified and sorted into three size classes (body size < 2.5 × 5, 5 × 10, and up to 10 × 20 mm). Tissue powder from each size category was extracted individually and pooled based on tissue weight to simulate samples that were not sorted by biomass (“Unsorted”). Additionally, size fractions were pooled so that each specimen contributed approximately equal amounts of biomass (“Sorted”). Mock samples were amplified using four different DNA metabarcoding primer sets targeting the Cytochrome c oxidase I (COI) gene. Sorting taxa by size and pooling them proportionately according to their abundance lead to a more equal amplification of taxa compared to the processing of complete samples without sorting. The sorted samples recovered 30% more taxa than the unsorted samples at the same sequencing depth. Our results imply that sequencing depth can be decreased approximately fivefold when sorting the samples into three size classes and pooling by specimen abundance. Even coarse size sorting can substantially improve taxa detection using DNA metabarcoding. While high‐throughput sequencing will become more accessible and cheaper within the next years, sorting bulk samples by specimen biomass or size is a simple yet efficient method to reduce current sequencing costs.     

Enrichment of progenitor cells from human bone marrow by flow cytometry

Mononuclear cells, harvested from fresh human bone marrow specimens by density gradient separation, were suspended in phosphate buffered saline and analyzed by flow cytometry in terms of the forward and right angle scattering of the incident light. The rectilinear distribution, obtained by plotting the intensity of light scattered in the forward and right angle directions, contained three regions of interest in which the percentage of cells (Mean ± standard deviation) with respect to the total was as follows: Region 1: 17.6±9.9; region 2: 5.3±1.4; region 3: 71.7±9.4. Cells from each region were sorted by flow cytometry and plated in semi-solid agar containing cell conditioned medium supportive of myeloid colony formation. Cells from region 2 contained the majority of progenitor cells that gave rise to such colonies at a plating efficiency that rose in proportion to the extent by which the region 2 cells in samples was increased through sorting. This increase in plating efficiency was 6 to 43 fold. Thus, region 2 of the cytometric distribution of cells from normal, unstained human bone marrows was a good source of myeloid progenitor cells.     

Apical cell surface expression of rat dipeptidyl peptidase IV in transfected Madin-Darby canine kidney cells.

Dipeptidyl peptidase IV (DPPIV) is a type II membrane glycoprotein that is predominantly localized to the apical plasma membrane in various epithelial cells. In order to understand in more detail the biogenesis and sorting of DPPIV, the cDNA for rat DPPIV was inserted into a mammalian plasmid expression vector so that DPPIV expression was driven by a control region composed of the SV40 early promoter region fused to the enhancer of the Rous sarcoma virus. Madin-Darby canine kidney cells transfected with this construct were found to express the DPPIV protein. In these transfected cells, the majority of DPPIV was present on the apial cell surface. This observation suggests that the information for apical surface localization is inherent in the DPPIV molecule itself and that this sorting information is decipherable in the epithelial cells of a different species. DPPIV is transported efficiently from the endoplasmic reticulum to the Golgi apparatus as assessed by pulse-chase experiments. Furthermore, evidence is presented which suggests that the majority of DPPIV is sorted intracellularly to the apical cell surface. The same protein has, however, been reported to be sorted by an indirect pathway through transcytosis from the basolateral to the apical cell surface in hepatocytes (Bartles, J.R., Feracci, H., M., Stinger, B., and Hubbard, A.L. (1987) J. Cell Biol. 105, 1241-1251). This study suggests that the same protein can take two different pathways in different cell types for its correct apical cell surface localization.     

Efficient enrichment of undifferentiated GFR alpha 1+ spermatogonia from immature rat testis by magnetic activated cell sorting

Spermatogonial stem cells (SSCs) are a documented source for adult multipotent stem cells. Thus, the isolation of SSCs is of great interest. However, the isolation of spermatogonia from mammalian testes is difficult because of their low total numbers and the lack of well-characterized cell surface markers. Glial-cell-derived neurotrophic factor family receptor alpha-1 (GFRα1) is expressed on undifferentiated mouse spermatogonia (including SSCs) and plays a crucial role, in rodents, for the maintenance of SSCs mediated by the Sertoli cell product GDNF. The present study has aimed to optimize the sorting efficiency and total cell yield of magnetic activated cell sorting (MACS) with anti-GFRα1 antibodies. Because of the technical limitations intrinsic to the magnetic columns, various sorting setups and strategies were compared. Use of Mini-MACS (MS) columns for single cell suspensions from 7-day-old rat testes resulted in a three-fold enrichment of GFRα1-positive cells in sorted fractions versus presorted fractions. However, with this method, only 1.77% of cells loaded onto the column were recovered in the sorted fraction. A sequential two-step sorting approach did not improve this poor yield. We therefore evaluated cell separation by using larger volume Midi-MACS (LS) columns. Enrichment of GFRα1-positive cells in sorted fractions was four-fold, and 14.5% of cells loaded onto the column were directed to the sorted fraction. With this method, approximately half of all GFRα1-positive cells present in the sample were found in the sorted fraction. We conclude that GFRα1 serves as a suitable surface marker for the enrichment of rat spermatogonia, and that the large-volume Midi-MACS separation system is superior to the routinely used small-volume Mini-MACS separation system. This work was financially supported by startup funds from the University Münster, NIH grant U54 HD 008610, Center grant, project 1 (to S.S.), a doctoral scholarship from the Ernst Schering Research Foundation (to K.G.), and a Young Investigator Grant from the Lance Armstrong Foundation (to J.E.).