Organelle Compartments Research Articles

NIPA1(SPG6), the Basis for Autosomal Dominant Form of Hereditary Spastic Paraplegia, Encodes a Functional Mg2+ Transporter

Mutations in the NIPA1(SPG6) gene, named for "nonimprinted in Prader-Willi/Angelman" has been implicated in one form of autosomal dominant hereditary spastic paraplegia (HSP), a neurodegenerative disorder characterized by progressive lower limb spasticity and weakness. However, the function of NIPA1 is unknown. Here, we show that reduced magnesium concentration enhances expression of NIPA1 suggesting a role in cellular magnesium metabolism. Indeed NIPA1 mediates Mg2+ uptake that is electrogenic, voltage-dependent, and saturable with a Michaelis constant of 0.69+/-0.21 mM when expressed in Xenopus oocytes. Subcellular localization with immunofluorescence showed that endogenous NIPA1 protein associates with early endosomes and the cell surface in a variety of neuronal and epithelial cells. As expected of a magnesium-responsive gene, we find that altered magnesium concentration leads to a redistribution between the endosomal compartment and the plasma membrane; high magnesium results in diminished cell surface NIPA1 whereas low magnesium leads to accumulation in early endosomes and recruitment to the plasma membrane. The mouse NIPA1 mutants, T39R and G100R, corresponding to the respective human mutants showed a loss-of-function when expressed in oocytes and altered trafficking in transfected COS7 cells. We conclude that NIPA1 normally encodes a Mg2+ transporter and the loss-of function of NIPA1(SPG6) due to abnormal trafficking of the mutated protein provides the basis of the HSP phenotype.

SUBCELLULAR DISTRIBUTION OF Ag, Cd, Co, Cu, Fe, Mn, Pb, AND Zn IN THE DIGESTIVE GLAND OF THE COMMON CUTTLEFISH SEPIA OFFICINALIS

The subcellular fractionation of the digestive gland cells of the common cuttlefish Sepia officinalis was performed to investigate the distribution of metals between organelles and cytosol and the different cytosolic fractions separated by gel-filtration chromatography. Total metal concentrations vary over 3 orders of magnitude, ranging from dwt for Pb to dwt for Zn. With the exception of Cd, Co, and Cu, metals were mostly bound to the organelles. Whereas no specific organelle compartment was found for Mn, Pb, and Zn, Fe was mainly associated with nucleus, brown body, and “boule” fraction (i.e., 52%) and 44% of the total Ag was contained in the lysosomal and mitochondria enriched fraction. The link of metals with hydrosoluble proteins in the cytosolic fraction was investigated at 254 and 280 nm. Direct relationship between cytosolic metal and metallothioneins could only be established for Ag and Cu, whereas Cd and Zn seem to mainly bind high (>70 kDa) and low (<4 kDa) molecular weight proteins.

The cytoskeleton maintains organelle partitioning required for single-cell C4 photosynthesis in Chenopodiaceae species.

Recently, three Chenopodiaceae species, Bienertia cycloptera, Bienertia sinuspersici, and Suaeda aralocaspica, were shown to possess novel C(4) photosynthesis mechanisms through the compartmentalization of organelles and photosynthetic enzymes into two distinct regions within a single chlorenchyma cell. Bienertia has peripheral and central compartments, whereas S. aralocaspica has distal and proximal compartments. This compartmentalization achieves the equivalent of spatial separation of Kranz anatomy, including dimorphic chloroplasts, but within a single cell. To characterize the mechanisms of organelle compartmentalization, the distribution of the major organelles relative to the cytoskeleton was examined. Examination of the distribution of the cytoskeleton using immunofluorescence studies and transient expression of green fluorescent protein-tagged cytoskeleton markers revealed a highly organized network of actin filaments and microtubules associating with the chloroplasts and showed that the two compartments in each cell had different cytoskeletal arrangements. Experiments using cytoskeleton-disrupting drugs showed in Bienertia and S. aralocaspica that microtubules are critical for the polarized positioning of chloroplasts and other organelles. Compartmentalization of the organelles in these species represents a unique system in higher plants and illustrates the degree of control the plant cell has over the organization and integration of multiorganellar processes within its cytoplasm.

Structural and functional compartmentalization in pollen tubes

Eukaryotic cellular functions are achieved by concerted activities in the cytosol and functions compartmentalized in the nucleus and other membrane-bound organelles. Moreover, the cytosol and nucleoplasm are populated with mega molecular ensembles that are specialized for different metabolic and biochemical processes. Pollen tubes are unique plant cells with a dramatic growth polarity. Tube growth is restricted to the tip and is supported by a polarized cytoplasmic organization. The apical region of elongating pollen tubes is a domain occupied exclusively by transport vesicles to support the secretion and endocytic activity needed for the rapid cell expansion at the apex. Larger organelles are predominantly segregated to the cytoplasm distal to the subapical region. Underlying the organelle compartmentalization is an elaborate actin cytoskeleton with distinct structural and dynamics properties at the tip, in the subapical region, and in the cytoplasm subtending it. Cytoplasmic domains with differential ionic conditions and spatially restricted localization of molecules in pollen tubes may also be important for regulating the polar cell growth process. The polarized cellular organization in pollen tubes drives an extremely efficient cell growth process that is responsive to extracellular signals, including directional cues. It may be an amplified framework of the cytoplasmic architecture that supports growth in other plant cell types that involves considerably more subtle and transient differential cell expansion.

Subcellular Localization of Mammalian Type II Membrane Proteins

Application of a computational membrane organization prediction pipeline, MemO, identified putative type II membrane proteins as proteins predicted to encode a single alpha-helical transmembrane domain (TMD) and no signal peptides. MemO was applied to RIKEN's mouse isoform protein set to identify 1436 non-overlapping genomic regions or transcriptional units (TUs), which encode exclusively type II membrane proteins. Proteins with overlapping predicted InterPro and TMDs were reviewed to discard false positive predictions resulting in a dataset comprised of 1831 transcripts in 1408 TUs. This dataset was used to develop a systematic protocol to document subcellular localization of type II membrane proteins. This approach combines mining of published literature to identify subcellular localization data and a high-throughput, polymerase chain reaction (PCR)-based approach to experimentally characterize subcellular localization. These approaches have provided localization data for 244 and 169 proteins. Type II membrane proteins are localized to all major organelle compartments; however, some biases were observed towards the early secretory pathway and punctate structures. Collectively, this study reports the subcellular localization of 26% of the defined dataset. All reported localization data are presented in the LOCATE database (http://www.locate.imb.uq.edu.au).

Gonadal steroid modulation of the diabetes ( db/ db) mutation-induced hyperlipometabolic, hypogonadal syndrome: Restoration of female reproductive tract cytochemical and structural indices

The gonadal steroids, 17- B-estradiol (E2) and progesterone (P), are recognized to stimulate cellular gluco- and lipo-metabolic compensatory cascades which counteract the deleterious influences of the diabetes ( db/ db) mutation (i.e., leptin membrane receptor defect) which promotes a progressive, hypercytolipidemia-induced premature involution of the female reproductive tract (FRT). The current studies define the therapeutic efficacy of E2 (1 μg/3.5 days) and P (1 mg /3.5 days) treatments (HRx) on utero–ovarian structural and cytochemical (gluco-/lipo-metabolic) maintenance, and the prevention of premature nuclear apoptosis and cytostructural disruption, following the expression of progressive db/ db-induced hypercytolipidemia. Control (normal: +/+ and +/?) and diabetes ( db/ db) genotype groups of 8-week-old (i.e., overt phase of the db/ db–hypogonadal syndrome) C57BL/KsJ mice were prepared for high resolution (HRLM) cytochemical and transmission electron (TEM) microscopic analysis of cytolipidemia and nuclear apoptosis (TUNEL-labeled 3′-DNA fragmentation) indices from uterine and ovarian secondary (early antral) follicular tissue samples. Compared to HRx controls, the db/ db mutation induced a dramatic increase in cytolipid vacuole volume and density within all ovarian follicular granulosa cells (GC) and uterine endometrial epithelial (UEE) layers. The co-localization of nuclear apoptotic 3′-DNA fragments within identified hyperlipidemic granulosa cells was coincident with the cytochemical and ultrastructural identification of lipid penetration through the nuclear envelope in db/ db mutants. P-HRx moderated the severity of db/ db-induced GC and UEE hypercytolipidemia, reducing the cytodensity of lipid vacuole accumulations and maintaining cytoplasmic organelle structure, organization, and nuclear membrane integrity. In contrast, E2-HRx resulted in a dramatic reduction in db/ db cytolipidemia in both ovarian GC and UEE tissue compartments. Following E2-HRx, UEE cells demonstrated non-pycnotic nuclear profiles, reduced nuclear apoptosis TUNEL-labeling, increased cytoplasmic organelle density profiles and a pronounced cytoplasmic cisternal expansion indicative of active cellular nutrient/metabolite trafficking. Ovarian follicular GC populations demonstrated minimal cytolipidemia, a restored cytoarchitecture with prominent organelle compartments and reduced TUNEL-indexed nuclear lipoapoptosis. These results are the first cytochemical and ultrastructural indications that P- and E2-HRx compensate for the genetic db/ db mutation-induced metabolic disturbances, which promote utero–ovarian hypercytolipidemia and the coincident nuclear lipoapoptosis culminating in the expressed diabetes hypogonadal syndrome. The capability of P-HRx to moderate the severity of utero–ovarian involution in db/ db mutants, and of E2-HRx to restore and maintain viable GC and UEE cyto-chemical and -structural indices under normoglycemic conditions, suggests that chronic, low-dose cyclic P- and E2-HRx stimulate cellular gluco- and lipo-metabolic cascades which compensate for the lack of leptin signaling in these single-gene, obese-Type II diabetic mutants. The compensatory endometabolic maintenance of utero–ovarian cellular and nuclear architecture suggests that the gluco- and lipo-metabolic disregulation may be therapeutically prevented or reversed, restoring reproductive tract cytointegrity and function, reducing the manifestation of hypogonadal reproductive sterility and db/ db compromise of the female reproductive tract.

Cytolipotoxicity-induced involution of the female reproductive tract following expression of obese ( ob/ ob) and diabetes ( db/ db) genotype mutations: progressive, hyperlipidemic transformation into adipocytic tissues

Both diabetes ( db/ db) and obese ( ob/ ob) single gene mutations induce a progressive, hyperglycemic–hyperinsulinemic endometabolic environment which promotes hypercytolipidemic, utero-ovarian involution in C57BL/KsJ mice. The progressive expression of the induced diabetes–obesity syndrome (DOS) results in female reproductive sterility and eventual organoatrophy. In order to define the intra-cytoplasmic alterations induced by the progressive cytolipidemia on cellular vitality, utero-ovarian tissue samples were collected from both control ( +/?) and littermate-matched ob/ ob or db/ db C57BL/KsJ mice at either 4 weeks (initial-onset DOS phase), 8 weeks (progressive, overt DOS phase), or 16 weeks (chronic-DOS phase) of age for cytolipid distribution analysis. All db/ db and ob/ ob mutant groups exhibited phenotypic obesity and systemic hyperglycemia–hyperinsulinemia relative to age-matched littermate +/? groups. In all db/ db and ob/ ob age groups, a progressive hypercytolipidemia was noted relative to +/? groups. When analyzed for lipid channeling, a progressive perinuclear mapping pattern of cytolipid distribution was noted. The primary locus of initial db/ db and ob/ ob cytolipid deposition was localized to the baso-polar regions in endometrial epithelia samples, or to the interstitium-thecal layer border of ovarian follicular compartments, during the initial-onset DOS phase. Progressively, intra-cytoplasmic lipid mobilization promoted a consistent perinuclear channeling of lipid vacuoles, ultimately isolating nuclear loci from the peripherally displaced cytoplasmic organelles within uterine epithelial layers. In db/ db and ob/ ob ovarian tissue samples, a progressive, gradient-related lipid infiltration of interstitial, thecal and, ultimately, granulosa cell layers promoted an enhanced rate of follicular-lipidemic atresia relative to +/? groups. In each tissue layer, the cytolipidemia promoted a dramatic perinuclear lipid-isolation barrier from intra-cytoplasmic organelle domains. With age-related exacerbation of the DOS syndrome, cytoplasmic nuclear-organelle displacement and lipoisolation resulted in cellular atresia, promoting the eventual utero-ovarian organoatrophy which characterized the chronic-DOS phase in db/ db and ob/ ob C57BL/KsJ mutants. These results indicate that the cytoinvolution associated with reproductive tract atrophy in these genetically mutant, diabetic–obese models is promoted by the disruption of the normal cytoarchitecture of utero-ovarian tissue layers induced by the progressive lipid sequestration, accumulation and ultimate isolation-induced disruption of intra-cellular organelle compartmentalization.

The application of mass spectrometry to membrane proteomics.

Membrane proteins perform some of the most important functions in the cell, including the regulation of cell signaling through surface receptors, cell-cell interactions, and the intracellular compartmentalization of organelles. Recent developments in proteomic strategies have focused on the inclusion of membrane proteins in high-throughput analyses. While slow and steady progress continues to be made in gel-based technologies, significant advances have been reported in non-gel shotgun methods using liquid chromatography coupled to mass spectrometry (LC/MS). These latter strategies facilitate the identification of large numbers of membrane proteins and modifications, and have the potential to provide insights into protein topology and orientation in membranes.

Protective functions of intracellular heat-shock protein (HSP) 70-expression in patients with severe sepsis.

Intracellular expression of heat-shock-protein 70 (HSP70) arose early in evolutionary development as a tool to protect cellular homeostasis. HSP70 detects proteins that are incorrectly folded or denatured. They form a complex with such proteins which can lead to correct folding, compartmentalization in organelles, or to proteolytic degradation. HSP70 also appears to protect proteins from degeneration. Intracellular HSP70-expression is induced by a wide variety of stimuli including heat, fever, hypoxia, oxygen radicals, endotoxins, cytokines, and heavy metal ions. Pre-emptive induction of HSP70-expression reduces organ dysfunction and mortality in animal models of sepsis.

Dual Targeting Property of the N-terminal Signal Sequence of P4501A1: TARGETING OF HETEROLOGOUS PROTEINS TO ENDOPLASMIC RETICULUM AND MITOCHONDRIA

Recent studies from our laboratory showed that the beta-naphthoflavone-inducible cytochrome P4501A1 is targeted to both the endoplasmic reticulum (ER) and mitochondria. In the present study, we have further investigated the ability of the N-terminal signal sequence (residues 1-44) of P4501A1 to target heterologous proteins, dihydrofolate reductase, and the mature portion of the rat P450c27 to the two subcellular compartments. In vitro transport and in vivo expression experiments show that N-terminally fused 1-44 signal sequence of P4501A1 targets heterologous proteins to both the ER and mitochondria, whereas the 33-44 sequence strictly functions as a mitochondrial targeting signal. Site-specific mutations show that positively charged residues at the 34th and 39th positions are critical for mitochondrial targeting. Cholesterol 27-hydroxylase activity of the ER-associated 1-44/1A1-CYP27 fusion protein can be reconstituted with cytochrome P450 reductase, but the mitochondrial associated fusion protein is functional with adrenodoxin + adrenodoxin reductase. Consistent with these differences, the fusion protein in the two organelle compartments exhibited distinctly different membrane topology. The results on the chimeric nature of the N-terminal signal of P4501A1 coupled with interaction with different electron transport proteins suggest a co-evolutionary nature of some of the xenobiotic inducible microsomal and mitochondrial P450s.

Selective Transfer of Calcium from an Acidic Compartment to the Mitochondrion of Trypanosoma brucei: MEASUREMENTS WITH TARGETED AEQUORINS

Organelle compartments are used by cells as reservoirs of exchangeable Ca2+ and as Ca2+ buffers. The following study uses recombinant aequorins (CYT-AEQ and MT-AEQ) to measure the dynamics of Ca2+ flux between organelles in procyclic forms of the pathogenic protozoan, Trypanosoma brucei. Emphasis is placed on the exchange between an acidic Ca2+ reservoir and the mitochondrion. The mammalian mitochondrial targeting sequence was functional in trypanosomes as determined by immunoblots, immunolocalizations, and the observation that MT-AEQ was in a compartment whose Ca2+ uptake was inhibited 82% with carbonyl cyanide p-trifluoromethoxyphenylhydrazone and KCN. The resting level of free calcium ion concentration in the mitochondrion ([Ca2+]mit) was slightly higher than that in the cytoplasm ([Ca2+]cyt) (400 +/- 50 nM and 290 +/- 40 nM, respectively). Melittin (125 nM) disrupted Ca2+ homeostasis by inducing Ca2+ influx across the plasma membrane. [Ca2+]cyt became slightly elevated to 410 +/- 100 nM, whereas [Ca2+]mit was selectively increased approximately 12-fold, with a broad peak at 4.8 +/- 1.9 microM. At the peak, the mitochondrion contained approximately three times more free Ca2+ than the cytosol. However, mitochondrial retention of the Ca2+ was transient. Similar selective transport into the mitochondrion was observed when Ca2+ efflux from an acidic compartment was induced with monensin (2 microg/ml) in the presence of 5 mM EGTA. [Ca2+]cyt was transiently elevated to 400 +/- 50 nM, whereas [Ca2+]mit was elevated to 3.3+/-1.3 microM. When cells were treated sequentially with monensin (2 microg/ml) and then melittin (200 nM), mitochondrial Ca2+ transport was normal. However, [Ca2+]cyt became elevated to a level that was 1.4-fold higher than with melittin alone. Overall, these data demonstrate that the trypanosome mitochondrion is not a reservoir of exchangeable Ca2+ in the resting cell. However, Ca2+ is selectively channeled to the mitochondrion from the plasma membrane or acidic Ca2+ storage compartment. Additionally, the acidic compartment contributes to maintenance of Ca2+ homeostasis in response to melittin.

Microfluorometric kinetic analysis of cathepsin B activity in single human thyroid follicular epithelial cells using image analysis and continuous monitoring.

The activity of a cysteine proteinase, cathepsin B (EC 3.4.22.1), was determined in unfixed single human thyroid follicular epithelial cells at room temperature using an image analysis system. The formation of the reaction product was monitored every minute by measuring the increasing fluorescence intensity of emitted light from a Schiff-base product formed by the substrate N-CBZ-ala-arg-arg-4-methoxy-2-naphthylamide and the coupling agent 5-nitrosalicylaldehyde. Non-specific fluorescent signals were eliminated by adjusting the video signal to zero using leupeptin, a specific inhibitor of cathepsins B, H and L. The enzyme activity was expressed as fluorescence intensity versus time. After a mean lag period of 5 min (range 4-8 min, n = 4), the enzyme activity increased linearly lasting on average 5 min (range 3-8 min), followed by a marked decrease in reaction rate for 3 min (range 1-4 min), and another linear increase for 11 min (range 8-14 min). The second linear part of the curve was not as steep as the first one. The reaction velocity recorded in individual granules resulted either in a biphasic curve or straight line, suggesting the presence of two distinct organelle compartments with differences in membrane permeability. It is concluded that human thyroid follicular epithelial cells in culture exhibit cathepsin B activity which can be monitored continuously by videomicrofluorometry without interference from non-specific fluorescence.

Hepatocyte fine structure during maturation and senescence

Aging is accompanied by a myriad of changes in cell structure, function, and composition. The fact that much of the information concerning age-related alterations in cellular morphology is qualitative precludes meaningful correlations with biochemical changes in order to enhance data interpretation. The mammalian liver has been subjected to both qualitative and quantitative evaluations of hepatocyte structure as a function of aging, i.e., development, maturation, and senescence. Although these data are characterized by considerable variability and, in some instances, blatant contradictions, there exists sufficient agreement in several parameters to permit a consensus in the inbred rat model. Certainly the volume of individual hepatocytes increases with age, and many of the organelle compartments reflect this change. While old rats exhibit a high incidence of polyploidy, there is no definitive evidence to demonstrate a concomitant increase in the binuclear hepatocyte index. Several specific hepatocellular organelles undergo changes in their relative volume or surface area that appear to correlate with functional alterations. The volume density of the lysosomal compartment enlarges significantly during senescence and is accompanied by increased activities of several constituent hydrolases. The hepatic concentration of smooth-surfaced endoplasmic reticulum declines markedly with aging, as does the yield of liver microsomes and the activities of several microsomal enzymes, e.g., mono-oxygenases and glucose-6-phosphatase. However, the responses of the majority of hepatocyte organelles to aging is varied and inconsistent based on the limited data currently available.

The Effect of Neutral Shade on the Structure of Mature, Sun-Acclimated Palisade Cells of Helianthus annuus L. II. 3-Day Exposure

Portions of mature leaves of Helianthus annuus var. Mammoth, field grown under full sunlight (FS), were shaded for 3 days at 40%, 25%, or 0% FS. Nonshaded leaf areas served as FS controls. The palisade cell structure of shaded and control leaf areas was analzyed stereologically. There were no detectable shade effects on the volume density values of the major organelle compartments except for a decrease in the starch compartment. The surface density values of chloroplast thylakoid membranes were significantly greater in shaded than in nonshaded chloroplasts at all levels of shading. The proportion of granal thylakoid surface area was greater than that for stromal thylakoids in shaded compared to nonshaded chloroplasts. There were no discernible shade effects on mean palisade cell volume. The total chlorophyll (chl)/mm3 of leaf and chl a/b ratios were lower in the shaded leaves than in the controls except for the full-shade sample (0%), which had higher chl a/b ratios than the control. Thylakoid packing ratios for palisade cells (mm2 thylakoid/mm2 leaf surface) were greater in the shade than in sun areas for 25% and 0% FS. Considering the overall response of thylakoid surface areas and changes in leaf anatomy, there was a greater light-gathering potential in the shaded half of the leaf. Although some shade response was observed in 40% FS (688 μmol s-1 m-2), the most pronounced responses occurred at 25% FS (347 μmol s-1 m-2). Thus, the threshold light level for shade response in sunflowers is near the latter value. These studies also show that cells within the same leaf respond independently to local irradiance levels.

Pre-lysosomal divergence of alpha 2-macroglobulin and transferrin: a kinetic study using a monoclonal antibody against a lysosomal membrane glycoprotein (LAMP-1).

We have used electron microscopic immunocytochemistry to compare the distribution of LAMP-1, a marker for lysosomal membranes, with the intracellular localization of alpha 2-macroglobulin (alpha 2-M) and transferrin at various time points after their endocytosis into cultured NIH 3T3 cells. The purposes of this study were (a) to determine how soon endocytic ligands reach lysosomal organelles, (b) to examine whether the intermediate endocytic vesicles gained lysosomal markers gradually or in a precipitous, discrete event, and (c) to examine the relationship, if any, between the pathway of recycling ligands and lysosomes. At early time points (0-5 min) after initiation of endocytosis, most structures containing alpha 2-M labeled with colloidal gold (receptosomes) were not labeled by anti-LAMP-1 detected using ferritin bridge or peroxidase immunocytochemistry. At late time points (greater than or equal to 15 min), the structures containing alpha 2-M (lysosomes) were strongly labeled by anti-LAMP-1. In contrast, transferrin that was directly labeled with ferritin was mostly located in LAMP-1-negative structures at all time points studied. The proportion of alpha 2-M-gold containing vesicles strongly labeled for LAMP-1 roughly paralleled the proportion of alpha 2-M-gold-containing structures positive for cytochemically detectable acid phosphatase. Our data indicate that ligands such as transferrin that are internalized through coated pits and receptosomes, but not delivered to lysosomes, do not traverse a lysosomal organelle compartment as marked by LAMP-1 content. Ligands such as alpha 2-M that are destined for lysosomal delivery reach a LAMP-1-positive organelle compartment only after they traverse LAMP-1-negative, non-lysosomal vesicles previously described as receptosomes.

Determination of connections between cellular structures using 3-d reconstruction from multiple-view stereopair images of thick sections

An important application for serial section reconstruction is determining whether a structure is composed of separate parts, or is continuous. Structures which are apparently separate on random sections may actually be continuous within the 3-D volume. The presence of connections between different types of organelle compartments within a cell should give important information about metabolic control of the cell. The determination of whether an organelle type is composed of multiple compartments, or actually exists as one or a few complexes, should also be of metabolic significance. The metabolic importance of some connections, such as gap junctions, between adjacent cells has already been well established.One method to prove that two membrane-bound structures connect is to trace the membrane continuously from one structure to the other. This is difficult when using thick sections because the membranes may be nearly invisible if they are at an oblique angle to the direction of view.

EFFECT OF SHORT‐TERM SHADING ON THE CYTOLOGY OF PALISADE TISSUE IN MATURE LEAVES OF THE SUNFLOWER HELIANTHUS ANNUUS

Mature sunflower leaves were exposed to partial shading (35 or 14% of normal sun) or darkness (0% of normal sun) for approximately 8 hr. During this period one‐half of each test leaf was shaded; the other half was used as a normal sun control. Palisade cell structure from both halves of each leaf was compared. Shading of leaves had little effect on organelle percent volume values (Vv) with exception of the starch compartment which decreased as shading increased. The surface to volume ratio (Sv) of the chloroplast thylakoids increased while the Sv of the mitochondrial membranes decreased as shading increased. Palisade cell volume did not change in shaded portions of the leaf, except in the fully shaded (dark) tissues where cell volume decreased. Changes in the actual volume of organelle compartments were strongly correlated with changes in cell volume. Thus a general osmotic response may account for some of the volume changes associated with differences in light intensity. Shading increased thylakoid surface areas 10–30% over the full sun controls. The ratio of stromal to granal thylakoid surface area remained constant in both the control and partially shaded samples. However, in darkened samples this ratio decreased as stromal membranes increased more than granal membranes. Changes observed in thylakoid surface areas associated with shading did not support thylakoid models which propose the interconversion of granal membranes to stromal membranes and vice versa.

Symbolic logic model of cellular adaptation

Human disease can be viewed as a set of quantitative alterations in existing metabolic pathways. We had previously constructed a symbolic logic model of quantitative organelle pathology, based upon the general pathology of growth disorders. The symbolic components of this model are cellular organelle compartments, measurements (number, surface, volume), and quantifiers (low, normal, high). These components allow one to deduce descriptive growth states, reaction patterns of cellular injury, and interorganelle homology patterns. Homologies have proved to be very useful in helping to elucidate difficult relationships in general pathology. In this report, we attempt to show that this usefulness also holds for quantitative organelle pathology. The types of cellular homology occurring in the nuclear-ergastoplasmic-mitochondrial-peroxisomal system reveal the evidence of existing interactions that can be determined rapidly and consistently by means of symbolic logic analysis. These cellular homology types allow us to draw conclusions regarding the capacity of the cell to adapt itself and the extent of cellular injury.

Polarized compartmentalization of organelles in growth cones from developing optic tectum.

We have used computer-assisted reconstructions of continuous serial sections to study the cytoplasmic organization of growth cones in vivo. Optic tecta from 6.25-6.5-d-old chicken embryos were quick-frozen and then freeze-substituted in acetone-osmium tetroxide or, for comparison, prepared by conventional fixation. Images of eight freeze-substituted and two conventionally fixed growth cones were reconstructed from aligned serial micrographs. After freeze-substitution, numerous lumenless membrane-bound sacs arrayed in multilamellar stacks appear to replace the abundant smooth endoplasmic reticulum found after chemical fixation. Microtubule fascicles progressively diverge from their typical fascicular organization in the initial segment of the growth cone and are absent in the varicosity and the more distal segment. Mitochondria, in contrast, are concentrated in the proximal segment of the varicosity; multilamellar stacks and endosome-like vacuoles are in the distal segment; and coated pits and vesicles are concentrated near the terminal filopodium, which is the most distal and organelle-poor domain of the growth cone. These observations suggest that dilation and fusion of the lumenless, membrane-bound sacs that occurs during chemical fixation give rise to the network of smooth endoplasmic reticulum. The three-dimensional reconstructions show that the cytoplasmic components of growth cones, including the membrane-bound sacs and multilamellar stacks revealed by freeze substitution, are polarized along the axis of these growth cones, which suggests that they have a role in recycling of membrane during elongation of the growth cone.

A MORPHOMETRIC ANALYSIS OF CYTOLOGICAL CHANGES DURING SPORE MATURATION IN THE MYXOMYCETE DIDYMIUM IRIDIS

Ultrastructure of spore maturation in the myxomycete Didymium iridis was investigated using morphometric analytical techniques. Changes in actual volume (μm3) and relative volume (Vv) of nuclei, autophagic vacuoles, mitochondria, microbodies, lipid droplets, and spore wall were described for spores in three stages of development. Stage I spores were newly formed, surrounded only by the cell membrane. Stage II spores were approximately 1 hr older than Stage I spores and possessed surface spines, but little if any additional wall material. Stage III spores were 24 hr old and possessed a fully formed, multilayered wall. The results of this study indicate that spore maturation in D. iridis is a multistep process involving a decrease in spore volume and coordinated changes in specific organelle compartments. From Stage I to Stage III, mean spore volume decreased by more than 50%. Percent volume data (Vv) showed that Stage I spores allocated volume equally to all measured organelles except microbodies and the spore wall, the latter of which had not yet begun to develop. By Stage II, only the nucleus and spore wall showed significant changes in Vv values, both increasing. In terms of actual volume, the nucleus, autophagic vacuole and spore wall increased by Stage II. Between Stages II and III the cell wall was the only component to increase in volume, all others decreased in volume. Our data indicate a close relationship between a decrease in organelle volume and an increase in cell wall volume in the Stage III spore. The autophagic vacuole and the cell wall dominated the volume of the Stage III spore while the remaining volume was allocated unequally to the other components.