Parity violation and the evolution of biomolecular homochirality

Parity violation at the level of terrestrial biopolymers, as seen in proteins, DNAs, and RNAs, and parity violation at the level of nuclear processes, as evident in longitudinally polarized beta-particles and parity-violating energy differences (PVEDs), are discussed and their fundamental importances are emphasized. Attempts to find a causal connection between the unique homochirality of biopolymers and parity violation at the nuclear level, and speculations that the former is a consequence of the latter, are reviewed. Consideration of all lines of evidence leads to the conclusion that there is no substantiation for such a causal connection, and that the two levels of parity violation are entirely independent of each other.     

Trafficking of immunoglobulin receptors in epithelial cells:signals and cellular factors

A typical feature of epithelial cells is the polarized distribution of their respective plasma membrane proteins. Apical and basolateral proteins can be sorted both in the trans-Golgi network and endosomes, or in both locations. Inclusion into basolateral carriers in the TGN requires the presence of distinct cytoplasmic determinants, which also appear to be recognized in endosomes. Inactivation of the basolateral sorting information leads to the efficient apical delivery, probably due to the unmasking of a recessive apical signal. Factors associated with the cytosolic face of organelles probably not only recognize these signals to mediate the inclusion of the proteins into the correct transport vesicles, but also target the carriers to the corresponding plasma membrane domain. Our interest has focused on analyzing at the molecular level how epithelial MDCK cells generate and maintain a polarized phenotype, taking advantage of immunoglobulin receptors to study the biosynthetic and endocytic pathways and the corresponding sorting events.     

Orientational changes of the absorbing dipole or retinal upon the conversion of rhodopsin to bathorhodopsin, lumirhodopsin, and isorhodopsin.

The orientational change of the absorbing dipole of the retinal chromophore in vertebrate rhodopsin (rhodo) upon photo-excitation to bathorhodopsin (batho), lumirhodopsin (lumi) and isorhodopsin (iso), has been studied by polarized absorption and linear dichroism measurements on magnetically oriented frog rod suspensions that were blocked at liquid nitrogen temperature. Both the azimuthal component delta theta and the polar component delta theta of the total angular change were studied in separate experiments. Delta theta was estimated from polarized absorption measurements on rods oriented transversally with respect to the analyzing beam. The data show unequivocally that upon the rhodo leads to batho transition, the dipole shifts out of the membrane plane by only few degrees; delta theta congruent to -3 degree. This azimuthal shift was nearly exactly reversed upon the batho leads to lumi decay. A very small shift (delta theta less than or equal to 1 degree) toward the membrane plane was observed upon a rhodo leads to iso conversion. The polar component delta theta of the angular shift was estimated by studying the photoreversion of linear dichroism induced by photo-excitation with polarized light in rods oriented parallel to the analyzing beam. Upon the rhodo leads to batho transition, ther was a shift delta theta = 11 +/- 3 degrees. The overall angular shift upon this first photo-exciting step, which corresponded to the isomerisation of retinal, was only delta omega = 11 +/- 3 degrees. This is smaller than what may be expected for a cis-trans isomerization of a retinal molecule with one end fixed, and different from what has been previously estimated by another group. These discrepancies are discussed.     

Myosin VI and Optineurin Are Required for Polarized EGFR Delivery and Directed Migration

The polarized trafficking of membrane proteins into the leading edge of the cell is an integral requirement for cell migration. Myosin VI and its interacting protein optineurin have previously been shown to operate in anterograde trafficking pathways, especially for the polarized delivery of cargo to the basolateral domain in epithelial cells. Here we show that in migratory cells ablation of myosin VI or optineurin inhibits the polarized delivery of the epidermal growth factor receptor (EGFR) into the leading edge and leads to profound defects in lamellipodia formation. Depletion of either myosin VI or optineurin, however, does not impair the overall ability of cells to migrate in a random migration assay, but it dramatically reduces directed migration towards a growth factor stimulus. In summary, we identified a specific role for myosin VI and optineurin in directionally persistent cell migration, which involves the polarized delivery of vesicles containing EGFR into the leading edge of the cell.     

Basolateral sorting of syntaxin 4 is dependent on its N-terminal domain and the AP1B clathrin adaptor, and required for the epithelial cell polarity

Generation of epithelial cell polarity requires mechanisms to sort plasma membrane proteins to the apical and basolateral domains. Sorting involves incorporation into specific vesicular carriers and subsequent fusion to the correct target membranes mediated by specific SNARE proteins. In polarized epithelial cells, the SNARE protein syntaxin 4 localizes exclusively to the basolateral plasma membrane and plays an important role in basolateral trafficking pathways. However, the mechanism of basolateral targeting of syntaxin 4 itself has remained poorly understood. Here we show that newly synthesized syntaxin 4 is directly targeted to the basolateral plasma membrane in polarized Madin-Darby canine kidney (MDCK) cells. Basolateral targeting depends on a signal that is centered around residues 24-29 in the N-terminal domain of syntaxin 4. Furthermore, basolateral targeting of syntaxin 4 is dependent on the epithelial cell-specific clathrin adaptor AP1B. Disruption of the basolateral targeting signal of syntaxin 4 leads to non-polarized delivery to both the apical and basolateral surface, as well as partial intercellular retention in the trans-Golgi network. Importantly, disruption of the basolateral targeting signal of syntaxin 4 leads to the inability of MDCK cells to establish a polarized morphology which suggests that restriction of syntaxin 4 to the basolateral domain is required for epithelial cell polarity.     

Interdependence of the Ypt/RabGAP Gyp5p and Gyl1p for recruitment to the sites of polarized growth

Gyp5p and Gyl1p are two members of the Ypt/Rab guanosine triphosphatases-activating proteins involved in the control of polarized exocytosis in Saccharomyces cerevisiae . We had previously shown that Gyp5p and Gyl1p colocalize at the sites of polarized growth and belong to the same complex in subcellular fractions enriched in plasma membrane or secretory vesicles. Here, we investigate the interaction between Gyp5p and Gyl1p as well as the mechanism of their localization to the sites of polarized growth. We show that purified recombinant Gyp5p and Gyl1p interact directly in vitro . In vivo , both Gyp5p and Gyl1p are mutually required to concentrate at the sites of polarized growth. Moreover, the localization of Gyp5p and Gyl1p to the sites of polarized growth requires the formins Bni1p and Bnr1p and depends on actin cables. We show that, in a sec6-4 mutant, blocking secretion leads to coaccumulation of Gyp5p and Gyl1p, together with Sec4p. Electron microscopy experiments demonstrate that Gyp5p is associated with secretory vesicles. Altogether, our results indicate that both Gyp5p and Gyl1p access the sites of polarized growth by transport on secretory vesicles. Two polarisome components, Spa2p and Bud6p, are involved in maintaining Gyp5p and Gyl1p colocalized at the sites of polarized growth.     

Dynamic Endothelial Cell Rearrangements Drive Developmental Vessel Regression

Patterning of functional blood vessel networks is achieved by pruning of superfluous connections. The cellular and molecular principles of vessel regression are poorly understood. Here we show that regression is mediated by dynamic and polarized migration of endothelial cells, representing anastomosis in reverse. Establishing and analyzing the first axial polarity map of all endothelial cells in a remodeling vascular network, we propose that balanced movement of cells maintains the primitive plexus under low shear conditions in a metastable dynamic state. We predict that flow-induced polarized migration of endothelial cells breaks symmetry and leads to stabilization of high flow/shear segments and regression of adjacent low flow/shear segments.     

Opposing roles for actin in Cdc42p polarization

In animal and fungal cells, the monomeric GTPase Cdc42p is a key regulator of cell polarity that itself exhibits a polarized distribution in asymmetric cells. Previous work showed that in budding yeast, Cdc42p polarization is unaffected by depolymerization of the actin cytoskeleton (Ayscough et al., J. Cell Biol. 137, 399-416, 1997). Surprisingly, we now report that unlike complete actin depolymerization, partial actin depolymerization leads to the dispersal of Cdc42p from the polarization site in unbudded cells. We provide evidence that dispersal is due to endocytosis associated with cortical actin patches and that actin cables are required to counteract the dispersal and maintain Cdc42p polarity. Thus, although Cdc42p is initially polarized in an actin-independent manner, maintaining that polarity may involve a reinforcing feedback between Cdc42p and polarized actin cables to counteract the dispersing effects of actin-dependent endocytosis. In addition, we report that once a bud has formed, polarized Cdc42p becomes more resistant to dispersal, revealing an unexpected difference between unbudded and budded cells in the organization of the polarization site.     

N-glycans and glycosylphosphatidylinositol-anchor act on polarized sorting of mouse PrP(C) in Madin-Darby canine kidney cells

The cellular prion protein (PrP(C)) plays a fundamental role in prion disease. PrP(C) is a glycosylphosphatidylinositol (GPI)-anchored protein with two variably occupied N-glycosylation sites. In general, GPI-anchor and N-glycosylation direct proteins to apical membranes in polarized cells whereas the majority of mouse PrP(C) is found in basolateral membranes in polarized Madin-Darby canine kidney (MDCK) cells. In this study we have mutated the first, the second, and both N-glycosylation sites of PrP(C) and also replaced the GPI-anchor of PrP(C) by the Thy-1 GPI-anchor in order to investigate the role of these signals in sorting of PrP(C) in MDCK cells. Cell surface biotinylation experiments and confocal microscopy showed that lack of one N-linked oligosaccharide leads to loss of polarized sorting of PrP(C). Exchange of the PrP(C) GPI-anchor for the one of Thy-1 redirects PrP(C) to the apical membrane. In conclusion, both N-glycosylation and GPI-anchor act on polarized sorting of PrP(C), with the GPI-anchor being dominant over N-glycans.     

Formation and functions of asymmetric microtubule organization in polarized cells

Although microtubules are known to be essential for chromosome segregation during cell division, they also play important roles in the regulation and function of cell polarity. Cell polarization is fundamental to appropriate tissue patterning and the regulation of cellular diversity during animal development. In polarized cells, microtubules are often organized asymmetrically along the polarity axis. Recent studies show that such asymmetry in microtubule organization is important to connect a cell's polarization with its polarized functions. In some cases, asymmetrically organized microtubule arrays themselves induce cell polarity. Here we present an overview of the mechanisms and functions of asymmetric microtubule organization and discuss the possible role of microtubule asymmetry in the symmetry-breaking that leads to cell polarization.     

肝细胞极性与膜蛋白分选的分子机制

肝细胞是高度特化的极性上皮细胞,细胞质膜蛋白的分选和极性转运对于肝细胞极性的建立与维持至关重要.首先,膜蛋白在内质网中合成,随后经高尔基体加工修饰,再由反面高尔基体进一步分选,最后通过膜泡运输等不同的机制分别转运到胆汁腔面或窦状隙面,行使其特殊的功能.近些年来,细胞内负责转运的细胞器和主要的分选信号已逐步被揭示.特别是循环内体也被证明参与了胆汁腔面和窦状隙面膜蛋白的极性分选和转运.肝细胞的极性一旦遭到破坏,将会引起胆汁分泌障碍以及其他肝脏功能的损伤,从而可能导致肝脏糖脂代谢紊乱,甚至丧失正常的生理功能.因此,深入研究肝脏细胞极性的形成与维持机制,将为多种肝脏疾病的预防和治疗寻找到新的方向和靶点,具有重要的理论和临床实践意义.     

Crag regulates epithelial architecture and polarized deposition of basement membrane proteins in Drosophila

The polarized architecture of epithelia relies on an interplay between the cytoskeleton, the trafficking machinery, and cell-cell and cell-matrix adhesion. Specifically, contact with the basement membrane (BM), an extracellular matrix underlying the basal side of epithelia, is important for cell polarity. However, little is known about how BM proteins themselves achieve a polarized distribution. In a genetic screen in the Drosophila follicular epithelium, we identified mutations in Crag, which encodes a conserved protein with domains implicated in membrane trafficking. Follicle cells mutant for Crag lose epithelial integrity and frequently become invasive. The loss of Crag leads to the anomalous accumulation of BM components on both sides of epithelial cells without directly affecting the distribution of apical or basolateral membrane proteins. This defect is not generally observed in mutants affecting epithelial integrity. We propose that Crag plays a unique role in organizing epithelial architecture by regulating the polarized secretion of BM proteins.     

Receptor clustering drives polarized assembly of ankyrin

Expression of the L1 family cell adhesion molecule neuroglian in Drosophila S2 cells leads to cell aggregation and polarized ankyrin accumulation at sites of cell-cell contact. Thus neuroglian adhesion generates a spatial cue for polarized assembly of ankyrin and the spectrin cytoskeleton. Here we characterized a chimera of the extracellular and transmembrane domains of rat CD2 fused to the cytoplasmic domain of neuroglian. The chimera was used to test the hypothesis that clustering of neuroglian at sites of adhesion generates the signal that activates ankyrin binding. Abundant expression of the chimera at the plasma membrane was not a sufficient cue to drive ankyrin assembly, since ankyrin remained diffusely distributed throughout the cytoplasm of CD2-neuroglian-expressing cells. However, ankyrin became highly enriched at sites of antibody-induced capping of CD2-neuroglian. Spectrin codistributed with ankyrin at capped sites. A green fluorescent protein-tagged ankyrin was used to monitor ankyrin distribution in living cells. Enhanced green fluorescent protein-ankyrin behaved identically to antibody-stained endogenous ankyrin, proving that the polarized accumulation of ankyrin was not an artifact of fixing and staining cells. We propose a model in which clustering of neuroglian induces a conformational change in the cytoplasmic domain that drives polarized assembly of the spectrin cytoskeleton.     

Role of actin and Myo2p in polarized secretion and growth of Saccharomyces cerevisiae

We examined the role of the actin cytoskeleton in secretion in Saccharomyces cerevisiae with the use of several quantitative assays, including time-lapse video microscopy of cell surface growth in individual living cells. In latrunculin, which depolymerizes filamentous actin, cell surface growth was completely depolarized but still occurred, albeit at a reduced level. Thus, filamentous actin is necessary for polarized secretion but not for secretion per se. Consistent with this conclusion, latrunculin caused vesicles to accumulate at random positions throughout the cell. Cortical actin patches cluster at locations that correlate with sites of polarized secretion. However, we found that actin patch polarization is not necessary for polarized secretion because a mutant, bee1Delta(las17Delta), which completely lacks actin patch polarization, displayed polarized growth. In contrast, a mutant lacking actin cables, tpm1-2 tpm2Delta, had a severe defect in polarized growth. The yeast class V myosin Myo2p is hypothesized to mediate polarized secretion. A mutation in the motor domain of Myo2p, myo2-66, caused growth to be depolarized but with only a partial decrease in the level of overall growth. This effect is similar to that of latrunculin, suggesting that Myo2p interacts with filamentous actin. However, inhibition of Myo2p function by expression of its tail domain completely abolished growth.     

Conformational transitions and vibronic couplings in acid ferricytochrome c: a resonance Raman study.

Resonance Raman spectral changes in ferricytochrome c as a function of pH between 6.7 and 1.0 are reported and the structural implication is discussed in terms of the "core-expansion" model advanced by L. D. Spaulding et al. [(1975) J. Am. Chem. Soc. 97, 2517]. The data are interpreted as indicating the iron in high-spin ferricytochrome c (at pH 2.0) with two water molecules as axial ligands lies in the plane of the porphyrin ring. At pH 1.0 there is a different high-spin form of cytochrome c which has an estimated iron out-of-plane distance of approximately 0.46 A. The effect of a monovalent anion at pH 2.0 is to produce a thermal spin mixture with predominant low-spin species. Excitation at approximately 620 nm in acid cytochrome c (pH 2.0) enhances only three depolarized ring vibrations at 1623, 1555, and 764 cm-1. Marked enhancement of depolarized modes relative to polarized and anomalously polarized modes is attributed to the vibronic coupling between porphyrin pi leads to pi and porphyrin pi leads to iron (dpi) charge-transfer states.     

The highly elongated Drosophila mechanosensory bristle - a new model for studying polarized microtubule function

Microtubules (MTs) are polar polymers that can facilitate asymmetric distribution of cell components, a process important for polarized cell growth. The highly elongated and polarized Drosophila mechnosensory bristle cytoplasm is filled with short MTs that constitute a significant component of the shaft cytoplasm. Inhibition of MT assembly affects biased axial growth in the bristle and highlights the importance of MTs for this process. We demonstrate that the vast majority of bristle MTs are organized in a polarized manner, minus-ends out. We also show that genetic disruption of the MT polarity affects the polar distribution of cell components and leads to an alteration in the biased axial shape of the bristle shaft. Thus, we suggest that the asymmetric organization of the MT population within the bristle cell shaft is necessary for the proper axial elongation of this cellular extension. We would also like to emphasize the benefits of using the bristle cell as a model for studying MTs and MT-binding proteins because changes to this cytoskeletal component result in easily recognized at the phenotypes.     

Coordination of cell polarity during Xenopus gastrulation

Cell polarity is an essential feature of animal cells contributing to morphogenesis. During Xenopus gastrulation, it is known that chordamesoderm cells are polarized and intercalate each other allowing anterior-posterior elongation of the embryo proper by convergent extension (CE). Although it is well known that the cellular protrusions at both ends of polarized cells exert tractive force for intercalation and that PCP pathway is known to be essential for the cell polarity, little is known about what triggers the cell polarization and what the polarization causes to control intracellular events enabling the intercalation that leads to the CE. In our research, we used EB3 (end-binding 3), a member of +TIPs that bind to the plus end of microtubule (MT), to visualize the intracellular polarity of chordamesoderm cells during CE to investigate the trigger of the establishment of cell polarity. We found that EB3 movement is polarized in chordamesoderm cells and that the notochord-somite tissue boundary plays an essential role in generating the cell polarity. This polarity was generated before the change of cell morphology and the polarized movement of EB3 in chordamesoderm cells was also observed near the boundary between the chordamesoderm tissue and na?ve ectoderm tissue or lateral mesoderm tissues induced by a low concentration of nodal mRNA. These suggest that definitive tissue separation established by the distinct levels of nodal signaling is essential for the chordamesodermal cells to acquire mediolateral cell polarity.     

Cell cycle progression and cell polarity require sphingolipid biosynthesis in Aspergillus nidulans

Sphingolipids are major components of the plasma membrane of eukaryotic cells and were once thought of merely as structural components of the membrane. We have investigated effects of inhibiting sphingolipid biosynthesis, both in germinating spores and growing hyphae of Aspergillus nidulans. In germinating spores, genetic or pharmacological inactivation of inositol phosphorylceramide (IPC) synthase arrests the cell cycle in G(1) and also prevents polarized growth during spore germination. However, inactivation of IPC synthase not only eliminates sphingolipid biosynthesis but also leads to a marked accumulation of ceramide, its upstream intermediate. We therefore inactivated serine palmitoyltransferase, the first enzyme in the sphingolipid biosynthesis pathway, to determine effects of inhibiting sphingolipid biosynthesis without an accumulation of ceramide. This inactivation also prevented polarized growth but did not affect nuclear division of germinating spores. To see if sphingolipid biosynthesis is required to maintain polarized growth, and not just to establish polarity, we inhibited sphingolipid biosynthesis in cells in which polarity was already established. This inhibition rapidly abolished normal cell polarity and promoted cell tip branching, which normally never occurs. Cell tip branching was closely associated with dramatic changes in the normally highly polarized actin cytoskeleton and found to be dependent on actin function. The results indicate that sphingolipids are essential for the establishment and maintenance of cell polarity via control of the actin cytoskeleton and that accumulation of ceramide is likely responsible for arresting the cell cycle in G(1).     

Retromer Regulates Postendocytic Sorting of β-Secretase in Polarized Madin-Darby Canine Kidney Cells

β-Amyloid (Aβ) peptides are generated from the successive proteolytic processing of the amyloid precursor protein (APP) by the β-APP cleaving enzyme (BACE or β-secretase) and the γ-secretase complex. Initial cleavage of APP by BACE leads into the amyloidogenic pathway, causing or exacerbating Alzheimer's disease. Therefore, their intracellular traffic can determine how easily and frequently BACE has access to and cleaves APP. Here, we have used polarized Madin-Darby canine kidney (MDCK) cells stably expressing APP and BACE to examine the regulation of their polarized trafficking by retromer, a protein complex previously implicated in their endosome-to-Golgi transport. Our data show that retromer interacts with BACE and regulates its postendocytic sorting in polarized MDCK cells. Depleting retromer, inhibiting retromer function, or preventing BACE interaction with retromer, alters trafficking of BACE, which thereby increases its localization in the early endocytic compartment. As a result, this slows endocytosis of apically localized BACE, promoting its recycling and apical-to-basolateral transcytosis, which increases APP/BACE interaction and subsequent cleavage of APP toward generation and secretion of Aβ peptides.     

In vivo polarization of IFN-gamma at Kupfer and non-Kupfer immunological synapses during the clearance of virally infected brain cells

Kupfer-type immunological synapses are thought to mediate intercellular communication between antiviral T cells and virally infected target Ag-presenting brain cells in vivo during an antiviral brain immune response. This hypothesis predicts that formation of Kupfer-type immunological synapses is necessary for polarized distribution of effector molecules, and their directed secretion toward the target cells. However, no studies have been published testing the hypothesis that cytokines can only form polarized clusters at Kupfer-type immunological synapses. Here, we show that IFN-gamma and granzyme-B cluster in a polarized fashion at contacts between T cells and infected astrocytes in vivo. In some cases these clusters were found in Kupfer-type immunological synapses between T cells and infected astrocytes, but we also detected polarized IFN-gamma at synaptic immunological contacts which did not form Kupfer-type immunological synaptic junctions, i.e., in the absence of polarization of TCR or LFA-1. This indicates that TCR signaling, which leads to the production, polarization, and eventual directed secretion of effector molecules such as IFN-gamma, occurs following the formation of both Kupfer-type and non-Kupfer type immunological synaptic junctions between T cells and virally infected target astrocytes in vivo.