Otto Baumann

Endoplasmic reticulum of animal cells and its organization into structural and functional domains.

The endoplasmic reticulum (ER) in animal cells is an extensive, morphologically continuous network of membrane tubules and flattened cisternae. The ER is a multifunctional organelle; the synthesis of membrane lipids, membrane and secretory proteins, and the regulation of intracellular calcium are prominent among its array of functions. Many of these functions are not homogeneously distributed throughout the ER but rather are confined to distinct ER subregions or domains. This review describes the structural and functional organization of the ER and highlights the dynamic properties of the ER network and the mechanisms that support the positioning of ER membranes within the cell. Furthermore, we outline processes involved in the establishment and maintenance of an anisotropic distribution of ER-resident proteins and, thus, in the organization of the ER into functionally and morphologically different subregions.

Calcium- and inositol polyphosphate-sensitivity of the calcium-sequestering endoplasmic reticulum in the photoreceptor cells of the honeybee drone

SummaryThe photoreceptor cells in the honeybee drone contain an elaborate Ca2+-sequestering endoplasmic reticulum (ER). We measured Ca-oxalate formation within the ER of permeabilized retinal slices with a microphotometer and studied the kinetics of Ca2+-uptake into the ER and the properties of Ins(1,4,5)P3-induced Ca2+-release.The ATP-dependent Ca2+-uptake mechanism has a high affinity for Ca2+: Uptake rate was half maximal at Ca2+free ≈ 0.6 μM.Addition of Ins(1,4,5)P3 caused a persistent depression of Ca-oxalate formation due to Ca2+ -release from the ER. The Ins(1,4,5)P3-dependent Ca2+-release mechanism has a high affinity (half maximal rate with 0.2 μM Ins(1,4,5)P3) and a high specificity for Ins(1,4,5)P3: Ins(2,4,5)P3 was 6 times, Ins(1,3,4,5)P4 was 15 times less potent in inducing Ca2+-release. 3 μM Ins(1,4)P2 had no detectable effect. The sensitivity for Ins(1,4,5)P3 was maximal between 280 nM and 1.6 μM Ca2+free and decreased at higher and lower Ca2+-concentrations.Our data show that the ER in invertebrate photoreceptor cells is an effective Ca2+ -sink and an Ins(1,4,5)P3-sensitive Ca2+-source. We support the idea (Payne et al. 1988) that the ER-network close to the photoreceptive membrane, the submicrovillar cisternae (SMC), are the light- and Ins(1,4,5)P3-sensitive Ca2+-stores.

Immunolocalization of Na,K-ATPase in blowfly photoreceptor cells

The Na,K-ATPase (sodium pump) plays a central role in the physiology of arthropod photoreceptors as it re-establishes gradients for Na+ and K+ after light stimulation. We have mapped the distribution of the Na,K-ATPase in the photoreceptors of the blowfly (Calliphora erythrocephala) by immunofluorescent and immunogold cytochemistry, and demonstrate that the distribution pattern is more complex than previously presumed. High levels of sodium pumps have been detected consistently in all photoreceptors R1-8 on the nonreceptive surface, but no sodium pumps are found on the microvillar rhabdomere. Within the nonreceptive surface of the cells R1-6, however, the sodium pumps are confined to sites juxtaposed to neighboring photoreceptor or glial cells; no sodium pumps have been detected on the parts of the nonreceptive surface exposed to the intra-ommatidial space. In R7 and R8, the sodium pumps are found over the entire nonreceptive surface. The cytoskeletal protein spectrin colocalizes with the sodium pumps suggesting that linkage of the pump molecules to the spectrin-based submembrane cytoskeleton contributes to the maintenance of the complex pattern of pump distribution.

Dopaminergic and serotonergic innervation of cockroach salivary glands: distribution and morphology of synapses and release sites

SUMMARY The paired salivary glands in the cockroach are composed of acini with ion-transporting peripheral P-cells and protein-secreting central C-cells, and a duct system for the modification of the primary saliva. Secretory activity is controlled by serotonergic and dopaminergic neurons, whose axons form a dense plexus on the glands. The spatial relationship of release sites for serotonin and dopamine to the various cell types was determined by anti-synapsin immunofluorescence confocal microscopy and electron microscopy. Every C-cell apparently has only serotonergic synapses on its surface. Serotonergic and dopaminergic fibres on the acini have their release zones at a distance of ∼0.5 μm from the P-cells. Nerves between acinar lobules may serve as neurohaemal organs and contain abundant dopaminergic and few serotonergic release sites. Some dopaminergic and serotonergic release sites reside in the duct epithelium, the former throughout the duct system, the latter only in segments next to acini. These findings are consistent with the view that C-cells respond exclusively to serotonin, P-cells to serotonin and dopamine, and most duct cells only to dopamine. Moreover, the data suggest that C-cells are stimulated by serotonin released close to their surface, whereas P-cells and most duct cells are exposed to serotonin/dopamine liberated at some distance.

THE ROLE OF ACTIN FILAMENTS IN THE ORGANIZATION OF THE ENDOPLASMIC RETICULUM IN HONEYBEE PHOTORECEPTOR CELLS

Close to the bases of the photoreceptive microvilli, arthropod photoreceptors contain a dense network of endoplasmic reticulum that is involved in the regulation of the intracellular calcium concentration, and in the biogenesis of the photoreceptive membrane. Here, we examine the role of the cytoskeleton in organizing this submicrovillar endoplasmic reticulum in honeybee photoreceptors. Immunofluorescence microscopy of taxol-stabilized specimens, and electron-microscopic examination of high-pressure frozen, freeze-substituted retinae demonstrate that the submicrovillar cytoplasm lacks microtubules. The submicrovillar region contains a conspicuous F-actin system that codistributes with the submicrovillar endoplasmic reticulum. Incubation of retinal tissue with cytochalasin B leads to depolymerization of the submicrovillar F-actin system, and to disorganization and disintegration of the submicrovillar endoplasmic reticulum, indicating that an intact F-actin cytoskeleton is required to maintain the architecture of this domain of the endoplasmic reticulum. We have also developed a permeabilized cell model in order to study the physiological requirements for the interaction of the endoplasmic reticulum with actin filaments. The association of submicrovillar endoplasmic reticulum with actin filaments appears to be independent of ATP, Ca2+ and Mg2+, suggesting a tight static anchorage.

Association of spectrin with a subcompartment of the endoplasmic reticulum in honeybee photoreceptor cells

The endoplasmic reticulum (ER) in honeybee photoreceptors is organized into structurally distinct subregions. The most prominent of these, the submicrovillar network of ER cisternae, is tightly associated with actin filaments. Electron microscopic techniques have demonstrated that the ER-associated actin filaments are regularly spaced at 60-80 nm and cross-bridged by filamentous structures. A polyclonal antibody against Drosophila alpha-spectrin has been used to examine the distribution of spectrin in the photoreceptors. On Western blots of bee retina, the antibody identifies a 260-kDa protein that exhibits biochemical and immunological properties characteristic of alpha-spectrin. Immunofluorescence microscopy has shown that alpha-spectrin codistributes with the submicrovillar ER but not with other ER subdomains. After cytochalasin-B-induced depolymerization of the ER-associated F-actin system, alpha-spectrin remains colocalized with the ER, indicating that alpha-spectrin is bound to the ER membrane. The F-actin/spectrin system associated with the submicrovillar ER may stabilize the shape of this ER subcompartment and may play a role in maintaining functional ER subregions.

The Golgi apparatus in honeybee photoreceptor cells: structural organization and spatial relationship to microtubules and actin filaments

Abstractu2002The architecture of the Golgi complex in honeybee photoreceptors has been analyzed by electron-microscopic techniques. The Golgi apparatus consists of several hundred individual stacks of cisternae dispersed throughout the soma of the photoreceptor cell. Two distinct subpopulations of Golgi stacks are distinguishable by their topographic features: (1) a dense row of Golgi stacks is aligned along the palisade-like cisternae of smooth endoplasmic reticulum backing the photoreceptive microvilli; (2) other Golgi stacks are scattered in the remainder of the cell body. The spatial relationship of Golgi stacks to microtubules and actin filaments has also been determined. Electron-microscopic examination of high-pressure-frozen freeze-substituted retinae reveals that Golgi stacks backing the submicrovillar endoplasmic reticulum reside in a cell area without microtubules, whereas the second subpopulation of Golgi stacks is embedded amidst microtubules. Labeling studies with several actin-specific probes, viz., rhodamine phalloidin, monoclonal anti-actin antibodies, and myosin fragments, provide evidence for a juxtaposition of the submicrovillar Golgi stacks to actin filaments. The Golgi membranes are thus ideally positioned to facilitate the transport of Golgi-derived material toward the microvilli along actin filaments.

Submembrane cytoskeleton of pigmented glial cells, primary pigment cells and crystalline cone cells in the honeybee compound eye

SummaryThe organization of the submembrane cytoskeleton of non-photoreceptive, accessory cells in the honeybee compound eye was examined using light-microscopic (phallotoxin labeling, immunohistochemistry) and electron-microscopic (decoration with myosin fragments) techniques. The crystalline cone cells contain numerous peripheral actin filaments oriented longitudinally with antiparallel polarity. Bundles of microtubules lie under the plasma membrane of primary pigment cells, in close apposition to the crystalline cone; they are interspersed with only a few actin filaments. Pigmented glial cells (secondary pigment cells) contain a two-dimensional filament/particle web lining their entire plasma membranes. Both filamentous actin and α-spectrin are localized within the cortex of these cells, indicating that they are web components. The results demonstrate that the three cell types contain different cortical cytoskeletons, implying different functional properties.

IMMUNOLOCALIZATION OF A PUTATIVE UNCONVENTIONAL MYOSIN ON THE SURFACE OF MOTILE MITOCHONDRIA IN LOCUST PHOTORECEPTORS

Abstractu2002Light stimulation of locust (Schistocerca gregaria) photoreceptors results in an actin-dependent translocation of mitochondria towards the photoreceptive microvilli and an antagonistic movement of endoplasmic reticulum towards the cell body. Using immunocytochemical techniques, we have tried to identify myosin-like motors that may drive the light-induced organelle motility. A monoclonal antibody against the motor domain of Acanthamoeba myosin identifies a prominent 110-kDa protein on Western blots of locust retina. Cross-reactivity with two polyclonal anti-myosin antibodies and a monoclonal anti-myosin-I-antibody, together with ATP-dependent binding to actin filaments, provides evidence that the 110-kDa protein is an unconventional myosin. By indirect immunofluorescence, the 110-kDa protein has been localized to both photoreceptors and pigment cells within the retina. In the photoreceptor cells, the 110-kDa protein is bound to the surface of mitochondria. This putative unconventional myosin may thus be a motor protein involved in the light-induced translocation of mitochondria in photoreceptors.

DISTRIBUTION OF NA+,K+-ATPASE IN PHOTORECEPTOR CELLS OF INSECTS

Light stimulation of insect photoreceptors causes opening of cation channels and an inward current that is partially carried by Na+ ions. There is also an efflux of K+ ions upon photostimulation. Na+ and K+ gradients across the photoreceptor membrane are reestablished by the activity of the enzyme Na+,K(+)-ATPase. About two-thirds of the total amount of ATP consumed in response to a light stimulus is attributed to the activity of this ion pump, demonstrating the importance of this enzyme for photoreceptor function. Insect photoreceptor cells are polarized epithelial cells; their plasma membrane is organized into two domains having a distinct morphology, molecular composition, and function. The visual pigment rhodopsin and the molecular components of the transduction machinery are localized in the rhabdomere, an array of densely packed microvilli, whereas Na+,K(+)-ATPase resides in the nonrhabdomeric membrane. Comparative immunolocalization studies on compound eyes of diverse insect species have demonstrated subtle variations in the distribution patterns of Na+,K(+)-ATPase. These may be accounted for by differences in the mechanisms responsible for Na+,K(+)-ATPase positioning.