Flow cytometry as a strategy to study the endosymbiosis of algae in Paramecium bursaria

BACKGROUND: The stable symbiotic association between Paramecium bursaria and algae is of interest to study such mechanisms in biology as recognition, specificity, infection, and regulation. The combination of algae-free strains of P. bursaria, which have been recently established by treating their stocks of green paramecia with herbicide paraquat (Hosoya et al.: Zool Sci 12: 807-810, 1995), with the cloned symbiotic algae isolated from P. bursaria (Nishihara et al.: Protoplasma 203: 91-99, 1998), provides an excellent clue to gain fundamental understanding of these phenomena. METHODS: Flow cytometry and light microscopy have been employed to characterize the algal cells after they have been released from the paramecia by ultrasonic treatment. Algal optical properties such as light scattering and endogenous chlorophyll fluorescence intensity have been monitored for symbiotic and free-living strains, and strains at stages of interaction with a host. RESULTS: Neither algal morphology nor chlorophyll content has been found to be altered by sonication of green paramecia. This fact allows to interpret in adequate degree changes in the optical properties of symbiont that just has been released from the association with a host (decreased forward light scatter and chlorophyll fluorescence signals). Optical characterization of both symbiotic and free-living algal strains with respect to their ability to establish symbioses with P. bursaria showed that chlorophyll content per cell volume seems to be a valuable factor for predicting a favorable symbiotic relationship between P. bursaria and algae. CONCLUSIONS: Flow cytometry combined with algae-free paramecia and cloned symbiotic algae identifies algal populations that may be recognized by host cells for the establishment of symbioses.     

Chemical Modification Causes Similar Change in Dependence on Water Activity of Chymotrypsin Hydration and Catalysis in Hexane

Amino groups in alpha-chymotrypsin were reacted with pyromellitic anhydride, introducing 17 to 32 additional carboxyl groups. This modification causes a major change in the water adsorption isotherm of the lyophilized protein powder. Little water is bound by the modified enzyme at water activity (a_w) below 0.35, but it shows increased water binding at a_w over 0.5. This correlates with a similar change in the a_w dependence of the catalytic activity of the enzyme powder suspended in hexane, with a much steeper increase in activity of the modified chymotrypsin.     

Phosphorylation at threonine-18 in addition to phosphorylation at serine-19 on myosin-II regulatory light chain is a mitosis-specific event

BACKGROUND: Cell division is an inevitable and vitally indispensable event in cell life, when the nucleus and cytoskeleton undergo profound reorganization. Cytoplasmic division (cytokinesis) is known to occur immediately after the end of nuclear division, when the nuclear envelope breaks down, and chromosomes condense and segregate, but its driving mechanism remains enigmatic. Myosin, particularly myosin-II, is thought to be required for cytokinesis as a force-generating element, the activity of which is mainly regulated through phosphorylations on its 20-kDa regulatory light chains (RLCs). MATERIALS AND METHODS: Multiparameter flow cytometric analysis was performed on fixed HeLa S3 cells (suspension culture cells) sequentially stained with the polyclonal antibody (termed PP1) against both phosphorylated sites (serine-19 and threonine-18) on the RLC, and with propidium iodide for DNA. "Positive" cells were sorted, followed by their microscopic examination. Fluorescence microscopy was employed to visualize the cell-cycle-dependent distribution of immunolabeled diphosphorylated RLCs in both HeLa S3 and adherent HeLa cells. RESULTS AND CONCLUSIONS: Doubly phosphorylated myosin RLCs were highly expressed in mitotic cells, suggesting the positive regulatory role of diphosphorylation in the redistribution of RLCs between daughter cells and then in cytokinesis. The increased immunofluorescence signal from the phosphorylated forms of RLC, together with flow cytometry, provides a clue with which to investigate the mechanisms governing the function of nonmuscle myosins during various cell motile events, including cytokinesis.     

Accumulation of oxacarbocyanine dyes with different alkyl chain length in bone marrow cells and hepatocytes

A study was made of the inclusion of fluorescent probe H-510 based on 3,3′-dialkyloxacarbocyanine bromide (where alkyl is ethyl, nonyl, or octadecyl) into cells of different types. Alkyl chain length (C2, C9, or C18) was found to largely determine the accumulation dynamics and mechanism. Similar spectral characteristics for all probe types in bone marrow cells were found by microfluorimetry, suggesting insertion of dye molecules irrespective of their lipophilicity into micelle-like structures formed probably by cell phospholipids. Spectroscopy data indicate interaction of 3,3′-diethyloxacarbocyanine bromide (H-510/C2) and 3,3-dinonyloxacarbocyanine bromide (H-510/C9) dyes in hepatocytes with a less polar microenvironment (nonpolar and low-polar lipids that constitute a significant part of the total content of cell lipids). The fluoresccence maximum of long-chain dye H-510/C18 in hepatocytes is shifted to the short-wavelength region and strictly coincides with the fluorescence maximum of the probe in an albumin solution. It is not excluded that inclusion of the probe into cells occurs via endocytosis upon its binding to surface proteins.     

Dynein is a motor for nuclear rotation while vimentin IFs is a “brake”

The positioning of the nucleus is achieved by two interconnected processes, anchoring and migration, both of which are controlled by cytoskeleton structures. Rotation is a special type of nuclear motility in many cell types, but its significance remains unclear. We used a vimentin-null cell line, MFT-16, which shows extensive nuclear rotation to study the phenomenon in detail. By selective disruption of cytoskeletal structures and video-microscopic analysis, nuclear rotation was a microtubule-dependent process that F-actin partially impedes. The dynein–dynactin complex is responsible and inhibiting this motor by expression of a dominant negative mutant of its component P-150 completely stops it. Nuclear rotation is powered by dynein associated with the nuclear envelope along stationary microtubules, centrosomes remaining immobile. We confirmed that vimentin IFs inhibit nuclear rotation, and variant proteins of the mutated wild type gene for vimentin that lacked considerable fragments of the N- and C-terminal domains restored nuclear anchoring. Immunochemical analysis showed that these mutated IFs also bound plectin, arguing for a key role of this cytolinker protein in nuclear anchoring. It is proposed that this versatile machinery guarantees not only rotation and the correct location of a nucleus, but also its orientation in a cell.     

Spatiotemporal patterns of macrophage migration inhibitory factor (Mif) expression in the mouse placenta

Background  

Macrophage migration inhibitory factor (MIF) has special pro-inflammatory roles, affecting the functions of macrophages and lymphocytes and counter-regulating the effects of glucocorticoids on the immune response. The conspicuous expression of MIF during human implantation and early embryonic development also suggests this factor acts in reproductive functions. The overall goal of this study was to evaluate Mif expression by trophoblast and embryo placental cells during mouse pregnancy.