Normal B cells express 51p1-encoded Ig heavy chains that are distinct from those expressed by chronic lymphocytic leukemia B cells

51p1 is an allele of V(H)1-69 that frequently is expressed by chronic lymphocytic leukemia (CLL) B cells with little or no somatic mutation. The rearranged 51p1 genes expressed by CLL B cells have a distinctive use of D segments D3-3/DXP4 and D3-10/DXP'1, a favored use of J(H)6, and a longer third complementarity-determining region than the rearranged Ig genes used by CLL B cells that express V(H)1 genes other than V(H)1-69. We examined the 51p1-encoded Ig expressed by blood B cells of healthy donors. In contrast to the infrequent use of J(H)4 by 51p1-expressing CLL (e.g., 4%), 36% of the rearranged 51p1 sequences from normal blood B cells used J(H)4. Furthermore, the D segment use of the rearranged 51p1 sequences from normal blood B cells was not restricted, but reflected the D segment use of nonselected IgH of normal B cells. Finally, the mean length of the third complementarity-determining region for the 51p1 genes of normal blood B cells was 14.6 +/- 4.3 (SD) codons. This is significantly shorter than that noted for 51p1-expressing CLL B cells (18.8 +/- 3.2; p < 0.0001, n = 51). This study demonstrates that the 51p1-encoded IgH expressed in CLL are not representative of the 51p1-encoded IgH expressed by normal blood B cells, indicating that CLL B cells express IgH that are distinctive from those found in the normal adult blood B cell repertoire.     

The Roadblock light chains are ubiquitous components of cytoplasmic dynein that form homo- and heterodimers

The Roadblock/LC7 class of light chains associate with the intermediate chains at the base of the soluble dynein particle. In mammals, there are two Roadblock isoforms (Robl1 and Robl2), one of which (Robl2) is differentially expressed in a tissue-dependent manner and is especially prominent in testis. Here we define the alpha helical content of Robl and demonstrate using both the yeast two-hybrid system and in vitro biochemistry that Robl1 and Robl2 are capable of forming homo- and heterodimers. This is the first report of heterodimer formation by any cytoplasmic dynein component, and it further enlarges the number of potential cytoplasmic dynein isoforms available for binding specific cellular cargoes. In addition, we have generated an antibody that specifically recognizes Robl light chains and shows a 5-10 fold preference for Robl2 over Robl1. Using this antibody, we show that Robl is a ubiquitous cytoplasmic dynein component, being found in samples purified from brain, liver, kidney, and testis. Immunofluorescence analysis reveals that Robl is present in punctate organelles in rat neuroblastoma cells. In testis, Robl is found in Leydig cells, spermatocytes, and sperm flagella.     

Distinct cytoplasmic dynein complexes are transported by different mechanisms in axons

In neurons, cytoplasmic dynein is synthesized in the cell body, but its function is to move cargo from the axon back to the cell body. Dynein must therefore be delivered to the axon and its motor activity must be regulated during axonal transport. Cytoplasmic dynein is a large protein complex composed of a number of different subunits. The dynein heavy chains contain the motor domains and the intermediate chains are involved in binding the complex to cargo. Five different intermediate chain polypeptides, which are the result of the alternative splicing of the two intermediate chain genes, have been identified. We have characterized two distinct pools of dynein that are transported from the cell body along the axon by different mechanisms. One pool, which contains the ubiquitous intermediate chain, is associated with the membranous organelles transported by kinesin in the fast transport component. The other pool, which contains the other developmentally regulated intermediate chains, is transported in slow component b. The mechanism of dynein regulation will therefore depend on which pool of dynein is recruited to function as the retrograde motor. In addition, the properties of the large pool of dynein associated with actin in slow component b are consistent with the hypothesis that this dynein may be the motor for microtubule transport in the axon.     

The intraflagellar transport dynein complex of trypanosomes is made of a heterodimer of dynein heavy chains and of light and intermediate chains of distinct functions

Cilia and flagella are assembled by intraflagellar transport (IFT) of protein complexes that bring tubulin and other precursors to the incorporation site at their distal tip. Anterograde transport is driven by kinesin, whereas retrograde transport is ensured by a specific dynein. In the protist Trypanosoma brucei, two distinct genes encode fairly different dynein heavy chains (DHCs; ∼40% identity) termed DHC2.1 and DHC2.2, which form a heterodimer and are both essential for retrograde IFT. The stability of each heavy chain relies on the presence of a dynein light intermediate chain (DLI1; also known as XBX-1/D1bLIC). The presence of both heavy chains and of DLI1 at the base of the flagellum depends on the intermediate dynein chain DIC5 (FAP133/WDR34). In the IFT140^RNAi mutant, an IFT-A protein essential for retrograde transport, the IFT dynein components are found at high concentration at the flagellar base but fail to penetrate the flagellar compartment. We propose a model by which the IFT dynein particle is assembled in the cytoplasm, reaches the base of the flagellum, and associates with the IFT machinery in a manner dependent on the IFT-A complex.     

Antibodies to cytoplasmic dynein heavy chain map the surface and inhibit motility

Polyclonal antibodies have been raised against four 16 residue peptides with sequences taken from the C-terminal quarter of the human cytoplasmic dynein heavy chain. The sites are downstream from a known microtubule-binding domain associated with the "stalk" that protrudes from the motor domain. The antisera were assayed using bacterially expressed proteins with amino acid sequences taken from the human cytoplasmic dynein heavy chain. Every antiserum reacted specifically with the appropriate expressed protein and with pig brain cytoplasmic dynein, whether the protein molecules were denatured on Western blots or were in a folded state. But, whereas three of the four antisera recognized freshly purified cytoplasmic dynein, the fourth reacted only with dynein that had been allowed to denature a little. After affinity purification against the expressed domains, whole IgG molecules and Fab fragments were assayed for their effect on dynein activity in in vitro microtubule-sliding assays. Of the three anti-peptides that reacted with fresh dynein, one inhibited motility but the others did not. The way these peptides are exposed on the surface is compatible with a model whereby the dynein motor domain is constructed from a ring of AAA protein modules, with the C-terminal module positioned on the surface that interacts with microtubules. We have tentatively identified an additional AAA module in the dynein heavy chain sequence, which would be consistent with a heptameric ring.     

All of the protein interactions that link steroid receptor.hsp90.immunophilin heterocomplexes to cytoplasmic dynein are common to plant and animal cells

Both plant and animal cells contain high molecular weight immunophilins that bind via tetratricopeptide repeat (TPR) domains to a TPR acceptor site on the ubiquitous and essential protein chaperone hsp90. These hsp90-binding immunophilins possess the signature peptidylprolyl isomerase (PPIase) domain, but no role for their PPIase activity in protein folding has been demonstrated. From the study of glucocorticoid receptor (GR).hsp90.immunophilin complexes in mammalian cells, there is considerable evidence that both hsp90 and the FK506-binding immunophilin FKBP52 play a role in receptor movement from the cytoplasm to the nucleus. The role of FKBP52 is to target the GR.hsp90 complex to the nucleus by binding via its PPIase domain to cytoplasmic dynein, the motor protein responsible for retrograde movement along microtubules. Here, we use rabbit cytoplasmic dynein as a surrogate for the plant homologue to show that two hsp90-binding immunophilins of wheat, wFKBP73 and wFKBP77, bind to dynein. Binding to dynein is blocked by competition with a purified FKBP52 fragment comprising its PPIase domain but is not affected by the immunosuppressant drug FK506, suggesting that the PPIase domain but not PPIase activity is involved in dynein binding. The hsp90/hsp70-based chaperone system of wheat germ lysate assembles complexes between mouse GR and wheat hsp90. These receptor heterocomplexes contain wheat FKBPs, and they bind rabbit cytoplasmic dynein in a PPIase domain-specific manner. Retention by plants of the entire heterocomplex assembly machinery for linking the GR to dynein implies a fundamental role for this process in the biology of the eukaryotic cell.     

Stress-dependent relocalization of translationally primed mRNPs to cytoplasmic granules that are kinetically and spatially distinct from P-bodies

Cytoplasmic RNA granules serve key functions in the control of messenger RNA (mRNA) fate in eukaryotic cells. For instance, in yeast, severe stress induces mRNA relocalization to sites of degradation or storage called processing bodies (P-bodies). In this study, we show that the translation repression associated with glucose starvation causes the key translational mediators of mRNA recognition, eIF4E, eIF4G, and Pab1p, to resediment away from ribosomal fractions. These mediators then accumulate in P-bodies and in previously unrecognized cytoplasmic bodies, which we define as EGP-bodies. Our kinetic studies highlight the fundamental difference between EGP- and P-bodies and reflect the complex dynamics surrounding reconfiguration of the mRNA pool under stress conditions. An absence of key mRNA decay factors from EGP-bodies points toward an mRNA storage function for these bodies. Overall, this study highlights new potential control points in both the regulation of mRNA fate and the global control of translation initiation.     

Circulating human B cells that express surrogate light chains display a unique antibody repertoire

Circulating human B cells that coexpress V-preB and conventional L chains (V-preB+L+ B cells) are a recently described subset of B cells that express Abs with features of self-reactivity. Initial analysis of V-preB+L+ B cells was limited to Ig-kappa and to the small, underused VH5 family. To determine whether Abs commonly expressed by V-preB+L+ B cells show similar features, we analyzed Ig H chains from three highly expressed VH families, VH1, VH3, and VH4, and Ig-lambda. We find that VH1 and VH3 Abs expressed by V-preB+L+ B cells resemble VH5 in that they display increased JH6 use, long CDR3s, and an increased frequency of D-D fusions. Abs in all three of these VH families also show skewed D reading frame use resulting in predominance of hydrophobic amino acids, which are counterselected in conventional B cells. Like Ig-kappa genes, the Ig-lambda genes in V-preB+L+ B cells show long CDR3s, but they differ from Ig-kappa genes in that they display no evidence of receptor editing. We conclude that a large number of H and L chain Abs expressed by V-preB+L+ B cells display features associated with self-reactive Abs.     

Aromatase-immunoreactive cells are present in mouse brain areas that are known to express high levels of aromatase activity

The transformation of testosterone into estradiol in the brain plays a key role in several behavioral and physiological processes, but it has been so far impossible to localize precisely the cells of the mammalian brain containing the relevant enzyme, viz., aromatase. We have recently established an immunohistochemical technique that allows the visualization of aromatase-immunoreactive cells in the quail brain. In this species, a marked increase in the optical density of aromatase-immunoreactive cells is observed in subjects that have been treated with the aromatase inhibitor, R76713 or racemic Vorozole. This increased immunoreactivity, associated with a total blockade of aromatase activity, has been used as a tool in the present study in which the distribution of aromatase-immunoreactive material has been reassessed in the brain of mice pretreated with R76713. As expected, the aromatase inhibitor increases the density of the immunoreactive signal in mice. Strongly immunoreactive cells are found in the lateral septal region, the bed nucleus of the stria terminalis, the central amygdala, and the dorso-lateral hypothalamus. A less dense signal is also present in the medial preoptic area, the nucleus accumbens, several hypothalamic nuclei (e.g., paraventricular and ventromedial nuclei), all divisions of the amygdala, and several regions of the cortex, especially the cortex piriformis. These data demonstrate that, contrary to previous claims, aromatase-immunoreactive cells are present in all brain regions that have been shown previously to contain high aromatase activity.     

Expression of distinct conformations of free HLA-Cw4 heavy chains in transfected neuroblastoma cells

Epitope mapping of HLA-Cw4 indicates that the two monoclonal antibodies (mAbs) L31 and M38, specific for beta 2-microglobulin (β₂m)-free HLA-C heavy chains, react preferentially with the KYK motif, located in the binding groove (α1 domain). Transfection of HLA-Cw4 cDNA into a neuroblastoma cell line, which normally expresses negligible HLA class I, resulted in the constitutive surface expression of molecules displaying different reactivities with the two mAbs. This cellular system was used to determine whether L31 and M38 recognize distinct conformations of β₂m-free HLA-C proteins, and to investigate their mechanism of expression. Interferon-γ greatly enhanced the expression of L31-reactive free chains, while abolishing that of M38-reactive molecules. The cytokine-induced expression of L31-reactive molecules was inhibited by anti-sense oligonucleotides specific for β₂m mRNA, while constitutive expression of L31-reactive molecules was only partially affected. Exogenous β₂m resulted in a reduction of constitutive L31 reactivity, and in a concomitant increase of M38 reactivity. These results indicate that: 1) at the cell surface, L31 and M38 react with two distinct conformations of HLA-Cw4 β₂m-free heavy chains, of which the L31-reactive conformation is the least folded; 2) the expression of both conformers can be modulated by endogenous or exogenous β₂m; and (3) L31-reactive molecules exposed at the cell surface are likely to derive from the dissociation of empty HLA-Cw4/β₂m complexes.     

Spnr, a murine RNA-binding protein that is localized to cytoplasmic microtubules

Previous studies in transgenic mice have established the importance of the 3' untranslated region (UTR) of the spermatid-specific protamine-1 (Prm-1) mRNA in its translational control during male germ cell development. To clone genes that mediate the translational repression or activation of the Prm-1 mRNA, we screened cDNA expression libraries made with RNA from pachytene spermatocytes and round spermatids, with an RNA probe corresponding to the 3' UTR of Prm-1. We obtained six independent clones that encode Spnr, a spermatid perinuclear RNA- binding protein. Spnr is a 71-kD protein that contains two previously described RNA binding domains. The Spnr mRNA is expressed at high levels in the testis, ovary, and brain, and is present in multiple forms in those tissues. Immunolocalization of the Spnr protein within the testis shows that it is expressed exclusively in postmeiotic germ cells and that it is localized to the manchette, a spermatid-specific microtubular array. Although the Spnr protein is expressed too late to be directly involved in the translational repression of Prm-1 specifically, we suggest that the Spnr protein may be involved in other aspects of spermatid RNA metabolism, such as RNA transport or translational activation.     

The Aspergillus cytoplasmic dynein heavy chain and NUDF localize to microtubule ends and affect microtubule dynamics

Cytoplasmic dynein is a multisubunit, minus end-directed microtubule motor that uses dynactin as an accessory complex to perform various in vivo functions including vesicle transport, spindle assembly, and nuclear distribution [1]. We previously showed that in the filamentous fungus Aspergillus nidulans, a GFP-tagged cytoplasmic dynein heavy chain (NUDA) forms comet-like structures that exhibited microtubule-dependent movement toward and back from the hyphal tip [2]. Here we demonstrate that another protein in the NUDA pathway, NUDF, which is homologous to the human LIS1 protein involved in brain development [3, 4], also exhibits such dynamic behavior. Both NUDA and NUDF are located at the ends of microtubules, and this observation suggests that the observed dynamic behavior is due to their association with the dynamic microtubule ends. To address whether NUDA and NUDF play a role in regulating microtubule dynamics in vivo, we constructed a GFP-labeled alpha-tubulin strain and used it to compare microtubule dynamics in vivo in wild-type A. nidulans versus temperature-sensitive loss-of-function mutants of nudA and nudF. The mutants showed a lower frequency of microtubule catastrophe, a lower rate of shrinkage during catastrophe, and a lower frequency of rescue. The microtubules in the mutant cells also paused longer at the hyphal tip than wild-type microtubules. These results indicate that cytoplasmic dynein and the LIS1 homolog NUDF affect microtubule dynamics in vivo.     

IL-4-producing gamma delta T cells that express a very restricted TCR repertoire are preferentially localized in liver and spleen.

IL-4-producing gamma delta thymocytes in normal mice belong to a distinct subset of gamma delta T cells characterized by low expression of Thy-1. This gamma delta thymocyte subset shares a number of phenotypic and functional properties with the NK T cell population. Thy-1dull gamma delta thymocytes in DBA/2 mice express a restricted repertoire of TCRs that are composed of the V gamma 1 gene product mainly associated with the V delta 6.4 chain and exhibit limited junctional sequence diversity. Using mice transgenic for a rearranged V gamma 1J gamma 4C gamma 4 chain and a novel mAb (9D3) specific for the V delta 6.3 and V delta 6.4 murine TCR delta chains, we have analyzed the peripheral localization and functional properties of gamma delta T cells displaying a similarly restricted TCR repertoire. In transgenic mice, IL-4 production by peripheral gamma delta T cells was confined to the gamma delta+9D3+ subset, which contains cells with a TCR repertoire similar to that found in Thy-1dull gamma delta thymocytes. In normal DBA/2 mice such cells represent close to half of the gamma delta T cells present in the liver and around 20% of the splenic gamma delta T cells.     

Mammalian cells express two VPS4 proteins both of which are involved in intracellular protein trafficking

The yeast Vps4 protein (Vps4p) is a member of the AAA protein family (ATPases associated with diverse cellular activities) and a key player in the transport of proteins out of a prevacuolar endosomal compartment. In human cells, we identified two non-allelic orthologous proteins (VPS4-A and VPS4-B) of yeast Vps4p. The human VPS4-A and VPS4-B proteins display a high degree of sequence identity to each other (80 %) and to the yeast Vps4 protein (59 and 60 %, respectively). Yeast cells lacking a functional VPS4 gene exhibit a temperature-sensitive growth defect and mislocalise a carboxypeptidase Y-invertase fusion protein to the cell surface. Heterologous expression of human VPS4 genes in vps4 mutant yeast strains led, in the case of human VPS4-A, to a partial and, in the case of human VPS4-B, to a complete suppression of the temperature-sensitive growth defect. The vacuolar protein sorting defect of vps4 mutant yeast cells was complemented completely by heterologous expressed human VPS4-B protein, and partially by the human VPS4-A protein. Expression of mutant human VPS4-A (E228Q) and VPS4-B (E235Q) proteins, harbouring single amino acid exchanges in their AAA domains, induced dominant-negative vacuolar protein sorting defects in wild-type yeast cells in both cases. Two-hybrid experiments suggest that the human VPS4-A and VPS4-B proteins can form heteromeric complexes, and subcellular localisation experiments indicate that both human VPS4 proteins associate with endosomal compartments in yeast. Based on these results, we conclude that both human VPS4 proteins are involved in intracellular protein trafficking, presumably at a late endosomal protein transport step, similar to the Vps4p in yeast.