mRNAs for alpha- and beta-tubulin and flagellar calmodulin are among those coordinately regulated when Naegleria gruberi amebae differentiate into flagellates

Three of four mRNAs that are specific to the differentiation of Naegleria gruberi amebae into flagellates (Mar, J., J. H. Lee, D. Shea, and C. J. Walsh, 1986, J. Cell Biol., 102:353-361) have been identified as coding for flagellar proteins. The products of these mRNAs, which are coordinately regulated during the differentiation, were identified by in vitro translation of hybrid-selected RNA followed by two-dimensional gel electrophoresis and antibody binding. Six cross-hybridizing clones complementary to a 1.7-kb RNA (class II) all selected mRNA that was translated into two alpha-tubulins. The principal in vitro product, alpha-1, comigrated with a cytoplasmic alpha-tubulin, while the minor product with a more acidic pI, alpha-2, comigrated with flagellar alpha-tubulin. While Naegleria flagellar alpha-tubulin was found to be acetylated based on its reaction with a monoclonal antibody specific to this form, we suggest that alpha-2 is not likely to arise due to acetylation in vitro but probably represents the product of a second alpha-tubulin gene. The class III clone, also complementary to a 1.7-kb RNA, selected beta-tubulin mRNA. In the course of this work it was found, using monoclonal antibodies to the alpha- and beta-subunits of tubulin, that Naegleria alpha-tubulin migrated faster than beta-tubulin on SDS-PAGE. The class IV clone, which hybridizes with a 0.5-kb RNA, selected an mRNA that was translated into a heat stable calcium-binding protein, flagellar calmodulin.     

The role of actin, actomyosin and microtubules in defining cell shape during the differentiation of Naegleria amebae into flagellates

Differentiation of Naegleria amebae into flagellates was used to examine the interaction between actin, actomyosin and microtubules in defining cell shape. Amebae, which lack microtubules except during mitosis, differentiate into flagellates with a fixed shape and a complex microtubule cytoskeleton in 120 min. Based on earlier models of ameboid motility it has been suggested that actomyosin is quiescent in flagellates. This hypothesis was tested by following changes in the cytoskeleton using three-dimensional reconstructions prepared by confocal microscopy of individual cells stained with antibodies against actin and tubulin as well as with phalloidin and DNase I. F-actin as defined by phalloidin staining was concentrated in expanding pseudopods. Most phalloidin staining was lost as cells rounded up before the onset of flagellum formation. Actin staining with a Naegleria-specific antibody that recognizes both F- and G-actin was confined to the cell cortex of both amebae and flagellates. DNase I demonstrated G-actin throughout all stages. Most of the actin in the cortex was not bound by phalloidin yet was resistant to detergent extraction suggesting that it was polymerized. The microtubule cytoskeleton of flagellates was intimately associated with this actin cortex. Treatment of flagellates with cytochalasin D produced a rapid loss of flagellate shape and the appearance of phalloidin staining while latrunculin A stabilized the flagellate shape. These results suggest that tension produced by an actomyosin network is required to maintain the flagellate shape. The rapid loss of the flagellate shape induced by drugs, which specifically block myosin light chain kinase, supports this hypothesis.     

Synthesis and assembly of the cytoskeleton of Naegleria gruberi flagellates

When Naegleria gruberi flagellates were extracted with nonionic detergent and stained by the indirect immunofluorescence method with AA-4.3 (a monoclonal antibody against Naegleria beta-tubulin), flagella and a network of cytoskeletal microtubules (CSMT) were seen. When Naegleria amebae were examined in the same way, no cytoplasmic tubulin-containing structures were seen. Formation of the flagellate cytoskeleton was followed during the differentiation of amebae into flagellates by staining cells with AA-4.3. The first tubulin containing structures were a few cytoplasmic microtubules that formed at the time amebae rounded up into spherical cells. The formation of these microtubules was followed by the appearance of basal bodies and flagella and then by the formation of the CSMT. The CSMT formed before the cells assumed the flagellate shape. In flagellate shaped cells the CSMT radiate from the base of the flagella and follow a curving path the full length of the cell. Protein synthetic requirements for the formation of CSMT were examined by transferring cells to cycloheximide at various times after initiation. One-half the population completed the protein synthesis essential for formation of CSMT 61 min after initiation of the differentiation. This is 10 min after the time when protein synthesis for formation of flagella is completed and 10-15 min before the time when the protein synthesis necessary for formation of the flagellate shape is completed.     

De novo formation of basal bodies during cellular differentiation of Naegleria gruberi: Progress and hypotheses

Under defined laboratory conditions, Naegleria gruberi undergo an amoeba-to-flagellate differentiation. During this differentiation, N. gruberi changes its shape from an amorphous amoeba to a regular shaped flagellate and forms de novo a flagellar apparatus, which is composed of two basal bodies, two flagella, a flagellar rootlet, and cytoplasmic microtubules. The entire process is accomplished within 2 h after initiation of differentiation and more than 95% of cells in the population undergo this differentiation. This rapid and synchronous differentiation of N. gruberi provides us with a unique system in which we can study the process of de novo basal body assembly. In this review, I summarize recent findings associated with de novo basal body assembly and propose a hypothesis to explain how N. gruberi assemble two basal bodies per cell, which is what happens in the majority of cells.     

Mitochondrial RNAs are abundant in the poly(A)+RNA population of early frog embryos

Mitochondrial sequences have been identified within a set of cloned complementary DNAs that had been copied from poly(A)⁺RNA of two embryonic stages of Xenopus laevis (Dworkin and Dawid, 1980, Dworkin and Dawid, 1980, Develop. Biol.76, 435–448 and 449–464). Mitochondrial sequences were found to be highly abundant in gastrula stage poly(A)⁺RNA sequences; in tadpole RNA their relative abundance is reduced severalfold. Mitochondrial sequences account for the most abundant poly(A)⁺RNA molecules in the gastrula population. The high abundance of mitochondrial RNA in early stages may be the consequence of the accumulation of large numbers of mitochondria in the egg.     

Two calmodulins in Naegleria flagellates: characterization, intracellular segregation, and programmed regulation of mRNA abundance during differentiation

Flagellates of Naegleria gruberi contain two calmodulins that differ in apparent molecular weight and intracellular location. Calmodulin-1, localized in flagella, has an apparent molecular weight of approximately 16,000, approximately the size of other protozoan calmodulins, whereas calmodulin-2, localized in cell bodies, is 15,300. Both proteins, purified, are calmodulins by several criteria, including Ca2+-dependent stimulation of calmodulin-dependent cyclic nucleotide phosphodiesterase and affinity for antibodies to vertebrate calmodulin. The finding of two calmodulins is unusual. Since the only known difference is apparent molecular weight, one calmodulin could be derived from the other, except that both calmodulins are synthesized in a wheat germ, cell-free system directed by RNA from differentiating Naegleria. Translatable mRNAs encoding calmodulins 1 and 2, not detected in amebas, appear and subsequently disappear concurrently during the 100-min differentiation of Naegleria from amebas to flagellates. Furthermore, these mRNAs increase and then decrease in abundance concurrently with those for flagellar tubulins, which suggests the possibility that the expression of the unrelated genes for calmodulin and tubulin may be under coordinate control during differentiation.     

Transient concentration of a gamma-tubulin-related protein with a pericentrin-related protein in the formation of basal bodies and flagella during the differentiation of Naegleria gruberi

The distribution of two proteins in Naegleria gruberi, N-gammaTRP (Naegleria gamma-tubulin-related protein) and N-PRP (Naegleria pericentrin-related protein), was examined during the de novo formation of basal bodies and flagella that occurs during the differentiation of N. gruberi. After the initiation of differentiation, N-gammaTRP and N-PRP began to concentrate at the same site within cells. The percentage of cells with a concentrated region of N-gammaTRP and N-PRP was maximal (68%) at 40 min when the synthesis of tubulin had just started but no assembled microtubules were visible. When concentrated tubulin became visible (60 min), the region of concentrated N-gammaTRP and N-PRP was co-localized with the tubulin spot and then flagella began to elongate from the region of concentrated tubulin. When cells had elongated flagella, the concentrated N-gammaTRP and N-PRP were translocated to the opposite end of the flagellated cells and disappeared. The transient concentration of N-gammaTRP coincided with the transient formation of an F-actin spot at which N-gammaTRP and alpha-tubulin mRNA were co-localized. The concentration of N-gammaTRP and formation of the F-actin spot occurred without the formation of microtubules but were inhibited by cytochalasin D. These observations suggest that the regional concentration of N-gammaTRP and N-PRP is mediated by actin filaments and might provide a site of microtubule nucleation for the assembly of newly synthesized tubulins into basal bodies and flagella.     

A transient species of poly(A)+ RNA detected by a myosin heavy chain cDNA probe in muscle cell culture during terminal differentiation

Primary cell cultures were prepared from breast muscles of 11 day 4 hour-embryonic chicks. Cytoplasmic RNAs were isolated from the cultured cells at various time intervals from day 3 to day 8. A [P32] DNA probe complementary to messenger RNA of myosin heavy chain was used to hybridize with the RNAs after gel electrophoresis. A transient species of polyadenylated RNA with a decreased mobility in electrophoresis was detected during a period of time when contractions of syncytial fibers were first observed.     

A study on the steady-state population of poly(A)+RNA during early development of Xenopus laevis

Autoantisera against rabbit testes and rabbit ejaculated spermatozoa have been used to study the appearance of surface autoantigens during spermatogenesis. Two distinct subclasses of autoantigens have been identified: an early subclass which first appears on pachytene spermatocytes and a late subclass which first appears on differentiating spermatids. These spermatids are just beginning to demonstrate migration of the nucleus and overlying acrosomal cap to the cell periphery and changes in nuclear shape. Some autoantigens of the early subclass do not appear on spermatozoa, but those that do are predominantly found over the acrosomal region. Autoantigens of the late subclass are predominantly found over the postacrosomal and middle-piece regions of the spermatozoon. It is suggested that morphological constraints during spermiogenesis may be responsible for the regional localization of the two subclasses.     

Relative amounts of newly synthesized poly(A)+ and poly(A)- messenger RNA during development of Xenopus laevis

The relative amounts of newly synthesized poly(A)⁺ and poly(A)^? mRNA have been determined in developing embryos of the frog Xenopus laevis. Polysomal RNA was isolated and fractionated into poly(A)⁺ and poly(A)^? RNA fractions with oligo(dT)-cellulose. In normal embryos the newly synthesized polysomal poly(A)⁺ RNA has a heterodisperse size distribution as expected of mRNA. The labeled poly(A)^? RNA of polysomes is composed mainly of rRNA and 4S RNA. The amount of poly(A)^? mRNA in this fraction cannot be quantitated because it represents a very small proportion of the labeled poly(A)^? RNA. By using the anucleolate mutants of Xenopus which do not synthesize rRNA, it is possible to estimate the percentage of mRNA which contains poly(A) and lacks poly(A). All labeled polysomal RNA larger than 4S RNA which does not bind to oligo(dT)-cellulose in the anucleolate mutants is considered presumptive poly(A)^? mRNA. The results indicate that about 80% of the mRNA lacks a poly(A) segment long enough to bind to oligo(dT). The poly(A)⁺ and poly(A)^? mRNA populations have a similar size distribution with a modal molecular weight of about 7 × 10⁵. The poly(A) segment of poly(A)⁺ mRNA is about 125 nucleotides long. Analysis of the poly(A)^? mRNA fraction has shown that it lacks poly(A)₁₂₅.     

5'-terminal structures of poly(A)+ cytoplasmic messenger RNA and of poly(A)+ and poly(A)- heterogeneous nuclear RNA of cells of the dipteran Drosophila melanogaster.

Structures at the 5′ terminus of poly (A)-containing cytoplasmic RNA and heterogeneous nuclear RNA containing and lacking poly(A) have been examined in RNA extracted from both normal and heat-shocked Drosophila cells. ³²P-labeled RNA was digested with ribonucleases T₂, T₁ and A and the products fractionated by a fingerprinting procedure which separates both unblocked 5′ phosphorylated termini and the blocked, methylated, “capped” termini, known to be present in the messenger RNA of most eukaryotes.Approximately 80% of the 5′-terminal structures recovered from digests of poly(A)-containing Drosophila mRNA are cap structures of the general form m⁷G^5′ppp^5′X(m)pY(m)pZp. With respect to the extent of ribose methylation and the base distribution, the 5′-terminal sequences of Drosophila capped mRNA appear to be intermediate between those of unicellular eukaryotes and those of mammals. Drosophila is the first organism known in which type 0 (no ribose methylations), type 1 (one ribose methylation), and type 2 (two ribose methylations) caps are all present. In contrast to mammalian cells, the caps of Drosophila never contain the doubly methylated nucleoside N⁶,2′-O-dimethyladenosine. Both purines and pyrimidines can be found as the penultimate nucleoside of Drosophila caps and there is a wide variety of X-Y base combinations. The relative frequencies of these different base combinations, and the extent of ribose methylation, vary with the duration of labeling. The large majority of poly(A)-containing cytoplasmic RNA molecules from heat-shocked Drosophila cells are also capped, but these caps are unusual in having almost exclusively purines as the penultimate X base.Greater than 75% of the 5′ termini of heterogeneous nuclear RNA (hnRNA) containing poly(A) and greater than 50% of the termini of hnRNA lacking poly (A) are also capped. Triphosphorylated nucleotides, common as the 5′ nucleotides of mammalian hnRNA, are rare in the poly(A)-containing hnRNA of Drosophila. The frequency of the various type 0 and type 1 cap sequences of cytoplasmic and nuclear poly (A)-containing RNA are almost identical. The caps of hnRNA lacking poly(A) are also quite similar to those of poly-adenylated hnRNA, but are somewhat lower in their content of penultimate pyrimidine nucleosides, suggesting that these two populations of molecules are not identical.     

Changes in hepatic RNA, poly(A)+RNA, and poly(A)-RNA during the acute phase response to inflammation

The steady state changes in total rat hepatic cytoplasmic RNA, poly(A)+ RNA and poly(A)-RNA were assessed in response to turpentine induced inflammation. From 18 to 24 h after injury, cytoplasmic RNA doubled, while poly(A)+ RNA peaked at 24 h, 3.5 times over control animals. Cell-free translation showed significant increases in messenger RNA levels beginning at 18 h. Gel electrophoresis of translation products revealed significant increases in several polypeptides and a decrease in others. Poly(A)-RNA from control and injured rats translated to an insignificant level and the electrophoretic gel patterns of their proteins were similar. Furthermore, no change had occurred in the 3' poly(A)-sequences during the course of inflammation.     

Characterization of the export of bulk poly(A)+ mRNA in Saccharomyces cerevisiae during the wine-making process

Ethanol stress affects the nuclear export of mRNA similarly to heat shock in Saccharomyces cerevisiae. However, we have little information about mRNA transport in actual alcoholic fermentation. Here we characterized the transport of mRNA during wine making and found that bulk poly(A)+ mRNA accumulated in the nucleus as fermentation progressed.