A new method called Allen Video-enhanced Contrast, Differential Interference Contrast (AVEC-DIC) microscopy is shown to be sufficiently sensitive to detect several new features of microtubule-related motility in the reticulopodial network of the foraminifer, Allogromia. The method takes advantage of the variable gain and offset features of a binary video camera to operate the DIC microscope under conditions highly favorable for video imaging, but in which the optical image is virtually invisible to the eye yet retains its full information when viewed by a suitable video camera. The improvements are made possible by setting a dé Senarmont compensator to lambda/9-lambda/4 at maximal working aperture of internally corrected planapochromatic objectives. Under these conditions, the offset feature of the video camera can reject so much stray light from the instrument and specimen that contrast compares favorably with that observed in high-extinction images, and polarizing rectifiers offer scarcely any advantage. Freed from the constraints of the light-limited conditions of DIC microscopy, video images can be recorded 60 times per second, or over 1,000 times the rate of photomicrographs at comparable magnifications under high-extinction conditions. Application of this method to the reticulopodial network of Allogromia has shown that cytoplasmic organelles are translocated only in contact with single microtubules or bundles of microtubules, and that these organelles fail to move when separated from microtubules. Microtubules themselves undergo both axial translatory ("sliding") and lateral "zipping and unzipping" movements that have been suggested to occur during mitosis and other biological processes.

Allogromia laticollaris, a benthic marine foraminifer, extends numerous trunk filopodia that repeatedly branch, anastomose, and fuse again to form the reticulopodial network (RPN), within which an incessant streaming of cytoplasmic particles occurs . The motion of the particles is saltatory and bidirectional, even in the thinnest filopodia detected by optical microscopy . Fibrils are visible by differential interference microscopy, and the RPN displays positive birefringence in polarized light. These fibrils remain intact after lysis and extraction of the RPN in solutions that stabilize microtubules (MTs) . Electron micrographs of thin sections through these lysed and stabilized cytoskeletal models reveal bundles of MTs. The RPNs of living Allogromia may be preserved by standard EM fixatives only after acclimatization to calcium-free seawater, in which the streaming is normal . The MTs in the RPN are typically arranged in bundles that generally lie parallel to the long axis of the trunk and branch filopodia. Stereo electron micrographs of whole-mount, fixed, and critical-point-dried organisms show that the complex pattern of MT deployment reflects the pattern of particle motion in both flattened and highly branched portions of the RPN . Cytoplasmic particles, some of which have a fuzzy coat, are closely associated with, and preferentially oriented along, either single MTs or MT bundles . Thin filaments (~5 nm) are also observed within the network, lying parallel to and interdigitating with the MTs, and in flattened terminal areas of the filopodia. These filaments do not bind skeletal muscle myosin S1 under conditions that heavily decorate actin filaments in controls (human blood platelets), and are^-20% too thin to be identified ultrastructurally as F-actin .For most of the motile phenomena exhibited by the various metazoan cell types, one can find couterparts within the Protista. Indeed, several protists have lent themselves to elegant biophysical (2, 3, 18) and, more recently, biochemical (16,33) investigations into all aspects of their motility. Bearing this in mind, we have undertaken a study of the motility ofAllogromia laticollaris (6), a marine benthic formainifer, as a possible model for the types of bidirectional particle transport observed in metazoan neurons. Allogromia extends slender filopodia that may reach a length of several millimeters . These filopodia repeatedly branch and fuse with one another, giving rise to a continuous anastomosing reticulopodial network (RPN). Within this network there is a constant bidirectional streaming of cytoplasm and particles (30). In the thinnest filopodia and in flattened areas of the RPN, particle motion is most easily observed, and here it is readily apparent that the motion is THE JOURNAL OF CELL BIOLOGY " VOLUME 90 JULY 1981 211-221 ©The Rockefeller University Press -0021-9525/81/07/0211/11 $1 .00 saltatory in character (1,(46)(47)(48) . The most slender of the filopodia detectable in the light microscope are <100 nm in diameter.The presence of struc...

A pollen-specific cDNA clone, Zinc13, has been isolated from a cDNA library constructed to poly(A) RNA from mature maize pollen. The cDNA as shown by primer extension analysis is a full-length copy of the mRNA. The cDNA has been sequenced and is 929 nucleotides in length plus a 47-nucleotide poly(A) tail. Putative polyadenylation signals are identifiable in the 3'-nontranslated region. The mRNA codes for a predicted polypeptide containing 170 amino acid residues and with a molecular mass of 18.3 kilodaltons. The hydropathy profile suggests a possible signal sequence on the amino terminus. A comparison of the nucleotide and deduced amino acid sequence with sequences in data banks has not shown homology to known molecules. In situ hybridizations using RNA probes show that the mRNA is located in the cytoplasm of the vegetative cell of the pollen grain and after germination is distributed throughout the pollen tube cytoplasm.

Lamellipodia have been induced to form within the reticulopodial networks of Allogromia laticollaris by being plated on positively charged substrata . Video-enhanced, polarized light, and differential interference contrast microscopy have demonstrated the presence of positively birefringent fibrils within these lamellipodia . The fibrils correspond to the microtubules and bundles of microtubules observed in whole-mount transmission electron micrographs of lamellipodia . Microtubular fibrils exhibit two types of movements within the lamellipodia : lateral and axial translocations . Lateral movements are often accompanied by reversible lateral associations between adjacent fibrils within a lamellipodium . This lateral association-dissociation of adjacent fibrils has been termed 'zipping' and 'unzipping' . Axial translocations are bidirectional . The axial movements of the microtubular fibrils can result in the extension of filopodia by pushing against the plasma membrane of the lamellipodia . Shortening, or complete withdrawal, of such filopodia is accomplished by the reversal of the direction of the axial movement .The bidirectional streaming characteristic of the reticulopodial networks also occurs within the lamellipodia . In these flattened regions the streaming is clearly seen to occur exclusively in association with the intracellular fibrils . Transport of both organelles and bulk hyaline cytoplasm occurs bidirectionally along the fibrils .Our previous studies (37) of the ultrastructure of the reticulopodial network of Allogromia demonstrated that the highly branched and anastomosed reticulopodium contains an extensive microtubule-based cytoskeleton. The microtubules were frequently bundled, forming a cytoskeletal system of far greater spatial organization than previously thought (22). By using sensitive (extinction factor of^-2,000) differential interference contrast optics, fibrillar elements were visualized within the most favorable areas of the reticulopodial networks of living organisms (37). The ultrastructural data indicated that these pseudopodial fibrils most certainly represented bundles of microtubules . The spatial distribution of microtubules within the reticulopodia reflected accurately the pattern of filopodial branching and anastomoses . This suggested that the microtubules might have a causal role in the formation and maintenance of pseudopodial form. In addition, the fibrils were oriented parallel to the direction ofthe cytoplasmic streaming, suggesting that they participated in some way in the intracellular transport process. 1668Recent advances in video-enhanced contrast microscopy (5, 6) have provided the means with which to study the cytoskeletal dynamics of living Allogromia. The video microscopic studies reported here were performed in part on flattened lamellipodial regions of the reticulopodial network of Allogromia . The results of the present studies clearly demonstrate that the microtubule-based cytoskeleton of the reticulopodial network is itself motile . The motility o...

A pollen-specific cDNA clone, Zinc13, has been isolated from a cDNA library constructed to poly(A) RNA from mature maize pollen. The cDNA as shown by primer extension analysis is a full-length copy of the mRNA. The cDNA has been sequenced and is 929 nucleotides in length plus a 47-nucleotide poly(A) tail. Putative polyadenylation signals are identifiable in the 3'-nontranslated region. The mRNA codes for a predicted polypeptide containing 170 amino acid residues and with a molecular mass of 18.3 kilodaltons. The hydropathy profile suggests a possible signal sequence on the amino terminus. A comparison of the nucleotide and deduced amino acid sequence with sequences in data banks has not shown homology to known molecules. In situ hybridizations using RNA probes show that the mRNA is located in the cytoplasm of the vegetative cell of the pollen grain and after germination is distributed throughout the pollen tube cytoplasm.

A new method is described for recording rapid processes of cell motility in polarized light. The Allen video-enhanced contrast (AVEC-POL) method of polarization microscopy achieves significant improvements in resolution, contrast, and the visibility of fine detail by a combination of novel adjustments to a standard (unrectified) polarizing microscope and video camera. Using the full working aperture of a high-power planapochromatic objective lens and compensator setting of lambda/9-lambda/4, visible images appear lacking in contrast. However, the same images viewed with an appropriate video camera equipped with an electronic offset adjustment can be made to appear with as much contrast as desired, revealing a significantly greater amount of fine detail in the image than can be seen by high extinction visual microscopy alone. At bias retardations between one-ninth and one-quarter wave, the diffraction anomaly observed near extinction disappears. Consequently, polarizing rectifiers are not required with the AVEC-POL method, and images previously requiring photographic exposures of around 20 seconds are sufficiently bright to be registered on the video monitor in 1/60 second. Using an intensity monitor, quantitative measurements of cellular birefringence can be retrieved from live or videotaped images displaying a linear relationship between contrast and phase retardation due to birefringence. The AVEC-POL method also renders accessible to polarized light analysis a number of objects that scatter or depolarize too much light to be studied by high extinction methods. The method is demonstrated on model objects and applied to the highly motile reticulopodial network of Allogromia laticollaris. Rapid motion in close association with microtubules can now be analyzed in greater detail at a significant reduction in the cost of recording.

A complex array of branching and anastomosing granular pseudopodia (reticulopodia) is thought to be a primary feature of the protistan phylum Granuloreticulosa. This essay summarizes the results of detailed structural and behavioral analyses of reticulopodia from representative species of ''naked,'' ''soft-shelled,'' and unilocular agglutinated Foraminifera. Promiscuous plasma membrane fusion, experimentally demonstrated here for the first time, together with the presence of tubulin polymorphs and a unique class of ellipsoid vesicles, form a suite of phenotypic characters that further define the suprageneric status of these protists. A theoretical framework for exploring the evolution of reticulopodia is also presented.

Cytoskeletal inhibitors were used as probes to test the involvement of microtubules and actin microfilaments in the development, motility, and shape maintenance of the pseudopodial networks (i e, reticulopodia) of the foraminifers Allogromia sp strain NF and Allogromia laticollaris. Agents that disassemble cytoplasmic microtubules (cold, colchicine, and nocodazole) arrest all movement but have variable effects on reticulopodial shape. Electron microscopy reveals a granulofibrillar matrix but few, if any, microtubules in these motility-arrested reticulopods. Allogromiids treated with cytochalasin B or D lose substrate adhesion and undergo dramatic changes in shape and motile behavior, highlighted by the coalescence of reticulopodial cytoplasm into irregularly shaped bodies with chaotic motility. Serial semithick sections of such preparations, viewed by high-voltage electron microscopy, document a striking rearrangement of microtubules within these cytochalasin-induced bodies. All aspects of cytochalasin-altered motility are completely inhibited by colchicine. Actin is present in reticulopodia, as determined by staining with rhodamine-phalloidin; this staining is not observed in cytochalasin-treated organisms. These data provide compelling evidence that microtubules are required for reticulopodial motility. An actin-based cytoskeleton is thought to play a role in maintaining shape, mediating pseudopod/substrate adhesion, and coordinating the various microtubule-dependent processes.