bluuebunny/crossref_metadata_2025 huggingface dataset:
Behavior of Dictyostelium amoebae is regulated primarily by the temporal dynamic of the natural cAMP wave
Authors: Wessels Deborah , Murray John , Soll David R.
Published: February 2005

The instantaneous velocity plots of Dictyostelium discoideum amoebae responding to natural waves and simulated temporal waves of cAMP with periods of 7 min are highly similar. This similarity has been used to deduce the dynamics of a natural wave crossing an amoeba, and the behavior of amoebae has been characterized during the different phases of a natural wave with a computer‐assisted dynamic image analyzing system. During the first ∼150 sec of the front of a natural wave, cells move persistently toward the aggregation center, with high instantaneous velocity and a decreased frequency of lateral pseudopod formation. During the last 30 sec of the front of the wave and the first 30 sec of the back of the wave, there is a “freeze” in cell shape and a dramatic depression in cell motility, pseudopod formation, and intracellular particle movement. During the last 180 sec of the back of the wave, there is a rebound in pseudopod formation, but it is random in direction and leads to no net cellular translocation. The data suggest that all of the behavior of a cell but orientation during the translocation phase is mediated by the temporal dynamics of the wave. The data also suggest that orientation toward the aggregation center occurs early in the front of the wave and that, once oriented, cells move in a blind fashion during the translocation phase. © 1992 Wiley‐Liss, Inc.
Expression of desmin cDNA in PtK2 cells results in assembly of desmin filaments from multiple sites throughout the cytoplasm
Authors: Mittal Balraj , Danowski Barbara A. , Sanger Jean M. , Sanger Joseph W.
Published: February 2005

The assembly of intermediate filaments into a cytoplasmic network was studied by microinjecting into the nuclei and cytoplasms of PtK2 cells, plasmids that contained a full length desmin cDNA and an RSV promoter. Immunofluorescence was used to monitor the expression of desmin and its integration into the cells' vimentin intermediate filament network. We found that the expressed desmin co‐localized with filaments of vimentin just as it does when fluorescently labelled desmin is microinjected into the cytoplasm of PtK2 cells. As early as two hours after microinjection of the plasmids, small discrete dots and short fragments of desmin could be detected throughout the cytoplasm of the cells. This initial distribution of desmin was superimposed on the filamentous pattern of vimentin in the cells. At 8 hours after microinjection of the plasmids, some of the desmin was present in long filaments that were coincident with vimentin filaments. By 18 hours, most of the desmin was in a filamentous network co‐localizing with vimentin. There was no indication that desmin assembly began in the perinuclear region and proceeded toward the cell periphery. In some cells, excessively high levels of desmin were expressed. In these cases, overexpression led to clumping of desmin filaments as well as to an accumulation of diffusely distributed desmin protein in the center of the cells. This effect was apparent at approximately 18 hours after introduction of the plasmid. The native vimentin filaments in such cells were also aggregated around the nucleus, colocalizing with desmin. The microtubule networks in all injected cells appeared normal; microtubules were extended in typical arrays out to the periphery of the cells. © 1992 Wiley‐Liss, Inc.
Characterization of the retraction response of goldfish retinal ganglion cell axons induced by monoclonal antibody 8A2 in vitro
Authors: Finnegan Sarah G. , Lemmon Vance P. , Koenig Edward
Published: February 2005

Retraction similar to that occurring spontaneously in senescent axonal fields of goldfish regenerating ganglion cell axons is reliably induced by monoclonal antibody (mAb) 8A2. The retraction response is characterized by transformation of the growth cone into a nodular motile mass, which undergoes retrograde translocation in conjunction with the contiguous column of axoplasm, generating evacuated distal strands. The growth cone‐to‐motile mass transformation involves a reorganization of F‐actin. In addition, the reorganization of F‐actin is a necessary antecedent for retrograde bulk translocation of axoplasm. Contractile tension contributes to compaction within the motile mass, while that within the column of distal axoplasm is oriented longitudinally and appears to contribute to bulk movement. As a derivative of the growth cone, the motile mass exhibits protrusive activities and a capacity to translocate independently when microtubules are partially disrupted. Apparent compressive forces cause buckling of microtubules in the adjacent segment which appear as elbow‐like protrusions. Cytochalasin D blocks mAb 8A2 induced retraction and immediately arrests retrograde translocation when it is in progress; however, neither nocodazole nor taxol blocks retraction. Phalloidin and immunofluorescence double labeling of retracted axons reveals that myosin 11, MLCK, and calmodulin co‐localize with dense F‐actin structures within the motile mass. These results suggest that microtubules play a subordinate, passive role, and that actomyosin interactions mediate the formation of the motile mass and the retraction response. Finally, axons grown on laminin exhibit a more robust retraction response than those grown on polylysine, implicating membrane‐cytoskeletal interactions as modulating factors. © 1992 Wiley‐Liss, Inc.
PDGF and neomycin induce similar changes in the actin cytoskeleton in human fibroblasts
Authors: Hedberg Karin M. , Bengtsson Torbjörn , Safiejko‐Mroczka Barbara , Bell Paul B. , Lindroth Margaretha
Published: February 2005

The addition of platelet‐derived growth factor (PDGF) to serum‐starved fibroblasts induces increased motility, formation of lamellipodia, increased ruffling activity, and actin ring structures associated with dorsal ruffles. Involvement of the phosphatidylinositol cycle (PI‐cycle) in these morphological changes was investigated by observing the effects of neomycin, an inhibitor of the PI‐cycle, on cultured human foreskin fibroblasts. The role of actin in the changes was investigated by using cytochalasin D (CD). Actin in detergent‐extracted cells was labelled with TRITC‐phalloidin and examined with fluorescence microscopy. Using PDGF and neomycin simultaneously potentiated lamellipodia formation, ruffling activity, as well as the number of cells with actin rings. Furthermore, neomycin by itself induced morphological changes similar to those induced by PDGF. Quantitation of actin rings showed dose and time dependency for PDGF and neomycin respectively, with a maximal number of cells containing rings after 15 min of exposure to either 3.5 mM neomycin or 10 ng PDGF/ml. Comparing the two substances, PDGF induced ring formation in a greater number of cells. These processes were inhibited by the presence of CD. PDGF‐ and neomycin‐induced changes in the actin cytoskeleton were also observed in human embryonic lung fibroblasts, human glial cells, and embryonic mouse fibroblasts, all of which are known to express PDGF‐receptors. In conclusion, the present study indicates that an increased turnover of the PI‐cycle is not essential for the changes in actin organization induced by PDGF. © 1993 Wiley‐Liss, Inc.
Myosin‐I in mammalian liver
Authors: Coluccio Lynne M. , Conaty Cathleen
Published: February 2005

Myosin‐I refers to a class of proteins with a molecular weight of approximately 110‐kDa, which have characteristics of conventional myosin but are unable to form filaments. Previous studies have implicated myosin‐I in motile cellular processes including cell migration and phagocytosis. Although the first example of myosin‐I in higher eukaryotes was the intestinal 110K‐calmodulin complex, which forms in microvilli the lateral links connecting the core bundle of actin filaments to the membrane, myosin‐I has now been shown to be a component of rat kidney and to be present in bovine adrenal gland and brain. We have now purified and characterized two polypeptides from rat liver which have several characteristics of the intestinal 110K‐calmodulin complex. Both liver polypeptides are solubilized with ATP and co‐elute on gel filtration with calmodulin. The polypeptides, of 110‐kDa and 130‐kDa, bind calmodulin in 1 mM EGTA. Both polypeptides bind to F‐actin in an ATP reversible fashion, and crosslink actin filaments. The purified polypeptides possess an actin‐activated Mg2+‐ATPase activity typical of brush border myosin‐I. A polyclonal antiserum directed against the chicken intestinal 110‐kDa polypeptide recognizes both rat liver polypeptides, whereas another serum recognizes the 130‐kDa but not the 110‐kDa rat liver polypeptide. Controlled proteolysis of the purified polypeptides with α‐chymotrypsin indicates that the two polypeptides are distinct but related. Immunofluorescence microscopy on isolated hepatocytes shows distribution of myosin‐I to be vesicular, distributed throughout the cytoplasm, but more concentrated near the nucleus. These data contribute new evidence by several functional criteria that multiple myosin‐I molecules are present in higher organisms and may coexist in a single cell type. © 1993 Wiley‐Liss, Inc.
Primary and secondary chick heart fibroblasts: Fast and slow‐moving cells show no significant difference in microtubule dynamics
Authors: Brown Denise A. , Warn Richard M.
Published: February 2005

Highly motile chick heart fibroblasts in primary culture (1° CHFs) gradually convert into much slower‐moving secondary (2°) cells. The polarized movement of the latter, but not the former, cell type has been found to be dependent on an intact microtubule (MT) network [Middleton et al., 1989, J. Cell Sci. 94:25–32]. To investigate the comparative stability of the MT networks of 1°s and 2°s, turnover was investigated by microinjection of biotin‐labeled brain tubulin to act as a reporter. MTs in both cell types were found to be very dynamic, with the MT networks effectively disassembled by about 30 min in 1° CHFs and 60 min in 2° CHFs, with mainly MT fragments remaining beyond these times. All MTs and fragments were found to have turned over by 1 h in 1° CHFs and 80 min in 2°s. Because 2° CHFs were found to be on average six times larger than 1°s, the difference in MT turnover time was considered largely due to the size difference. For both 1° and 2° cells, the more slowly turning over MTs were generally curly and perinuclear in distribution, resembling stable MTs in other systems, but they appeared significantly earlier in CHFs. However, no discrete subpopulations of slower turning over MTs were found to be associated with either the leading edges or the processes of either cell type. In addition, no major differences were identified in the patterns of modified α‐tubulin along the MTs or of MT cold or drug stability. It is concluded that MTs do not have a direct structural or skeletal function in maintaining a polarized 2° CHF cell shape, but rather play an ancillary role. © 1993 Wiley‐Liss, Inc.
Elastic properties of titin filaments demonstrated using a “freeze‐break” technique
Authors: Trombitás Károly , Pollack Gerald H. , Wright John , Wang Kuan
Published: February 2005

A “freeze‐break” technique (Trombitás, K.: Acta Biochim. Biophys. Hung. 6:419–427, 1971) and immunoelectron microscopy were used to study the elastic properties of titin filaments. Small bundles of freshly prepared rabbit psoas muscle fibers were quickly frozen and broken under liquid nitrogen to fracture sarcomeres in planes perpendicular to the filament axis, in each of various regions along the sarcomere. The still‐frozen specimens were thawed during fixation to allow elastic filaments to retract. The broken specimens were then labelled with monoclonal anti‐titin antibodies against an unique epitope in the I‐band.

The titin epitopes were normally positioned symmetrically about the Z‐line. However, in sarcomeres broken at the A‐I junction, the epitopes no longer remained symmetrical: the titin filaments in the broken half‐sarcomere retracted, independently of the thin filaments, forming a dense band just near the Z‐line. The retracted density apparently did not reach the Z‐line; retraction stopped at the level of the so‐called N1‐line. In sarcomeres broken at the Z‐line level, the titin filaments retracted in the opposite direction. In this case the titin epitope retracted all the way to the ends of the thick filaments.

It appears then that titin molecules form elastic filaments that are independent of thin filaments in most of the I‐band. Near the Z‐line, however, the titin filaments either have an inelastic domain or associate firmly with the thin filaments at the N1‐line level. © 1993 Wiley‐Liss, Inc.
Actin filament structure probed with monoclonal antibodies
Authors: Moncman Carole L. , Peng Isaac , Winkelmann Donald A.
Published: February 2005

The interaction of two monoclonal antibodies (mAbs) with actin has been characterized to map the epitopes defined by these mAbs and to determine the accessibility of these sites in the actin filament (F‐actin). Both mAbs react specifically with actin in radioimmunoassays and Western blot assays, and by immunoprecipitation. The location of the epitopes within the primary structure of actin has been determined using limited proteolysis of actin and Western blots, or using immunoprecipitation of truncated actin fragments synthesized in a cell free translation assay. Both mAbs bind to the C‐terminal fragment of actin (residues 68–375) produced by chymotrypsin cleavage. One epitope is further localized to a 9.9 kD peptide corresponding to residues 5–93. Therefore, the epitope defined by this mAb (2G11.4) lies between residues Lys68 and Glu93 of actin. The location of the other epitope was determined by immunoprecipitation of actin fragments synthesized in vitro. Removal of residues 356–365 from the C‐terminus of actin completely abolished the binding of mAb 4E3.adl. Therefore, this mAb defines an epitope that involves residues between Trp356 and Ala365. The accessibility of these epitopes in native F‐actin was determined with solution binding assays and characterized by immunoelectron microscopy. Monoclonal antibody 4E3.adl binds strongly to filaments, resulting in bundling or decoration of F‐actin depending on the valency of the mAb, and indicating that the epitope is readily accessible in F‐actin. In contrast, mAb 2G11.4 disrupts F‐actin structure, resulting in the formation of an amorphous immunoprecipitate. These results place constraints on models of actin filament structure. © 1993 Wiley‐Liss, Inc.
Characterization of fluorescently derivatized bovine tau protein and its localization and functions in cultured Chinese hamster ovary cells
Authors: Lu Qun , Wood John G.
Published: February 2005

Bovine brain tau protein was tagged with the fluorescent dye 5 (and 6)‐carboxy‐x‐rhodamine‐succinimidyl ester and the functional properties of the fluorescent analog were tested in vitro by kinetic measurement and SDS gel electrophoresis. X‐rhodamine tau was competent to bind to microtubules and promote microtubule assembly in vitro. Labeled tau was further characterized by microinjection of cultured Chinese hamster ovary (CHO) cells to study its intracellular distribution and potential new functions. X‐rhodamine tau incorporated rapidly into centrosomes within seconds after microinjection. It distinctly labeled the microtubule network as early as 5 to 10 minutes following microinjection. In addition, X‐rhodamine tau was transported into the nucleus and labeled the nucleolus specifically. Double labeling of the injected cells with DiC6(3) indicated that in some cases, fluorescent tau may associate with the endoplasmic reticulum. The concentrations of injected X‐rhodamine tau ranged from 1.7 to 5.0 mg/ml, yet distinct bundling of microtubules was not observed. Studies of nocodazole effects on the microtubules established that X‐rhodamine tau stabilized microtubules against depolymerization conditions. We conclude that this fluorescent analog of tau is associated with microtubules, the nucleolus, and other microtubule‐related structures in living cells, and is competent to stabilize microtubules against microtubule depolymerizing drug treatment. This approach provides a useful model system for the study of modified tau in neurodegenerative disease. © 1993 Wiley‐Liss, Inc.
Intracellular guanosine‐5′‐0‐(3‐thiotriphosphate) blocks chemotactic motility of Dictyostelium discoideum amoebae
Authors: Schlatterer Christina , Malchow Dieter
Published: February 2005

Starving amoebae of the cellular slime mold Dictyostelium discoideum react chemotactically towards the attractant cAMP. In this study, the effect of nonhydrolyzable analogs of GTP and GDP on the chemotactic behavior was analyzed with light microscopic techniques. Guanosine‐5′‐0‐(2‐thiotriphosphate) (GTPβS) or guanosine‐5′‐0‐(2‐thiodiphosphate) (GDPβS) was scrape‐loaded into the cytoplasm of cells, together with a fluorescent marker. Stimulation with a cAMP‐filled glass capillary revealed a reduced capacity of loaded cells to migrate to wards the capillary tip. Most cells still protruded filopods in the direction of the capillary tip, but full extension of pseudopods was inhibited in a dose‐dependent and reversible manner. This indicates that in the presence of the analogs, chemotactic sensing still occurs, and that a more distal step of the cascade of events leading to the formation of the pseudopod is impaired.

In cells loaded with the analogs together with the calcium indicator fura‐2, stimulation with 10 μM cAMP led to a transient change in the intracellular free calcium concentration ([Ca2+]i), which was detectable in 28% of the cells. Furthermore, large vacuoles were found containing high amounts of calcium. On the other hand, clamping of [Ca2+]i at low levels with 1,2‐bis(2‐aminophenoxy) ethane N,N,N′,N′‐tetraacetic acid (BAPTA) also inhibited motility, with neither filopods nor pseudopods formed.

The data suggest that chemotactic migratory activity involves GTP‐dependent processes that participate in the regulation of the Ca2+ homeostasis of the cell and in the regulation of membrane traffic that contributes to the directed locomotion. © 1993 Wiley‐Liss, Inc.
Localization of CapZ during myofibrillogenesis in cultured chicken muscle
Authors: Schafer Dorothy A. , Waddle James A. , Cooper John A.
Published: February 2005

Actin filaments undergo dramatic changes in their organization during myofibrilloenesis. In mature skeletal muscle, both CapZ and the barbed end of the actin filaments are located at Z‐discs. In vitro, CapZ binds the barbed end of actin filaments and prevents actin subunit addition and loss; CapZ also nucleates actin polymerization in vitro. Taken together, these properties suggest that CapZ may function to organize actin filaments during myofibrillogenesis. We report here that the amount of CapZ in myofibrils from adult chicken pectoral muscle is sufficient to “cap” each actin filament of the sacromere. Double inmmunofluorescence microscopy of skeletal muscle cells in culture was used to determine the spatial and temporal distributions of CapZ relative to actin, α‐actinin, titin, and myosin during myofibrilloenesis. Of particular interest was the assembly of CapZ at nascent Z‐discs in relation to the organization of actin filaments in nascent myofibrils. In myoblasts and young myotubes, CapZ was diffusely distributed in the cytoplasm. As myotubes matured, CapZ was initially observed in a uniform distribution along non‐striated actin filaments called stress fiber‐like structures (SFLS). CapZ was observed in a periodic pattern characteristic of mature Z‐discs along the SFLS prior to the appearance of a striated staining pattern for actin. In older myotubes, when actin was observed in a pattern characteristic of I‐bands, CapZ was distributed in a periodic pattern characteristic of mature Z‐discs. The finding that CapZ was assembled at nascent Z‐discs before actin was observed in a striated pattern is consistent with the hypothesis that CapZ directs the location and polarity of actin filaments during I‐band formation in skeletal muscle cells. The assembly of CapZ at nascent Z‐disc structures also was observed relative to the assembly of sarcomeric α‐actinin, titin, and thick filaments. Titin and myosin were observed in structures having the organization of mature sarcomeres prior to the appearance of CapZ at nascent Z‐discs. The distribution of CapZ and sarcomeric α‐actinin in young myotubes was not coincident; in older myotubes, both CapZ and α‐actinin were co‐localized at Z‐discs. In cardiac myocytes, CapZ was detected at Z‐discs and was distributed in a punctate pattern throughout the cytoplasm. CapZ also was co‐localized with A‐CAM and vinculin at cell‐cell junctions formed by the myocytes. © 1993 Wiley‐Liss, Inc.
Near‐UV radiation disrupts filamentous actin in lens epithelial cells
Authors: Rafferty Nancy S. , Zigman Seymour , McDaniel Thurma , Scholz Diane L.
Published: February 2005

Ultraviolet radiation in the near range (UVA) causes lens opacification and disrupts the actin cytoskeleton in rabbit and gray squirrel lenses. Changes were noted using transmission electron microscopy of tangential sections and rhodaminephalloidin fluorescence microscopy of epithelial whole mounts of irradiated and unirradiated lenses, and corresponded with gross cataract formation. Irradiated lenses lacked microfilament polygonal arrays at the inner surface of the apical plasma membrane (i.e., in the cell pole next to the lens fibers) in lens epithelia of both species; a condensed actin bundle was present instead. This bundle, and scattered small actin clumps in the cytoplasm, were identified by immunogold TEM, using a specific antibody and a secondary antibody conjugated with coloidal gold. Similar techniques showed breakdown of tubulin and vimentin, but after longer intervals than for the breakdown of actin. Generalized cytologic damage was also present in epithelial cells, but not in the underlying cortical lens fibers. Damage began to occur after 4 hr of irradiation and became more severe with increased exposure. Shielded controls remained clear, had normal cytology and polygonal arrays, and no clumping of actin filaments. © 1993 Wiley‐Liss, Inc.
Decreased levels of the high molecular weight subunit of neurofilaments and accelerated neurofilament transport during the recovery phase of 2,5‐hexanedione exposure
Authors: Pyle Sally J. , Amarnath Venkataraman , Graham Doyle G. , Anthony Douglas C.
Published: February 2005

The neurotoxicant 2,5‐hexanedione (HD) causes the accumulation of neurofilaments in the distal axon and an acceleration of neurofilament transport proximal to the site of their accumulation. It has been proposed that the acceleration of transport is due to the direct reaction of HD with neurofilament proteins and, conversely, that this acceleration is a secondary response of the axon to injury. The objective of this study was to determine whether the response of axons to HD intoxication includes acceleration of neurofilament transport. Pulse labeling was used to analyze neurofilament transport in age‐matched rats exposed to HD or PBS. The animals receiving HD were exposed either throughout the period of radiolabel transport, or prior to the pulse labeling of neurofilament proteins. If acceleration of the rate of neurofilament transport was due to the direct reaction of HD with proteins, then neurofilaments synthesized after the exposure period should travel at control rates, since these proteins would not have been exposed to the toxicant. After 28 days of transport, optic nerve proteins were examined using SDS‐PAGE, fluorography, and computerized densitometry. In both HD‐treated groups, neurofilament transport was accelerated relative to age‐matched control animals. In addition, the amount of NFH was decreased relative to other neurofilament subunits. The combination of accelerated transport and a diminished proportion of NFH is similar to the observations of neurofilament axonal transport during growth and development. These observations suggest that this persistent, secondary effect is a reparative response to injury that recapitulates axonal growth and development. © 1993 Wiley‐Liss, Inc.
Actin, α‐actinin, and vinculin are associated with septate junctions in insecta
Authors: Colombo Anita , Bonfanti Patrizia , Camatini Marina
Published: February 2005

Cytoskeletal elements associated with the smooth septate junctions linking the midgut columnar cells of Manduca sexta larvae (Insecta, Lepidoptera) were characterized. Myosin subfragment 1 decoration and immunostaining for actin demonstrated that the filaments associated with the septate junctions were constituted of actin. Moreover, using a combination of immunochemical and immunolocalization techniques, evidence is presented that α‐actinin, myosin II, and vinculin are localized close to the specialized plasma membranes. The insertion of microfilament bundles into submembranous F‐actin/α‐actinin/vinculin complexes, previously described in vertebrate junctions of adherens type, appears to be a more general organization, including the insect septate junction here examined. © 1993 Wiley‐Liss, Inc.
Smooth muscle myosin subfragment‐1 is a kinetic analogue for heavy meromyosin in the extended conformation
Authors: Drew Jean S. , White Marianne P. , Stein Leonard A.
Published: February 2005

The 10S→6S (Flexed→Extended) transition in smooth muscle myosin is related to increased ATPase activity, but there is controversy over whether the analogous 9S→7S transition in HMM is also associated with ATPase activity. We therefore studied the association of ionic strength, phosphorylation, and ATPase activity for HMM as compared to S1 which has no apparent flexed conformation. In addition, we performed both steady state and single turnover analyses, to control for artifacts due to multiple subfragment populations that might skew steady state results.

At low ionic strength where myosin and HMM are in the flexed conformation, HMM had a near zero ATPase activity while S‐1 had a high ATPase rate (0.07 s−1). At 400 mM ionic strength, where both myosin and HMM are in the extended conformation, S1 and HMM had the same ATPase rate (0.04 s−1). Phosphorylation did not affect S1 significantly, but shifted the HMM curve to higher rates at lower ionic strengths. Both steady state and single turnover experiments gave the same results, indicating that steady state results were not skewed by multiple subfragment populations. These data indicate that HMM has a conformation‐ATPase relation similar to that observed with myosin. Furthermore, these findings suggest that the S1 ATPase rate corresponds to that of HMM in the extended conformation. © 1993 Wiley‐Liss, Inc.
Differential distribution of glutamylated tubulin during spermatogenesis in mammalian testis
Authors: Fouquet Jean‐Pierre , Kann Marie‐Louise , Edde Bernard , Wolff Annie , Desbruyeres Elisabeth , Denoulet Philippe
Published: February 2005

The distribution of glutamylated tubulin has been analyzed in mammalian testis using the specific mAb GT335 by immunoelectron microscopy and immunoblotting. In spermatozoa of various species, immunogold labeling showed the presence of glutamylated tubulin in all of the microtubules of axoneme and centrioles, whereas the microtubule network of the spermatid manchette was unlabeled. In earlier germ cells, centriole was the only microtubule structure to be labeled. A similar distribution was observed using the anti‐acetylated tubulin antibody (6‐11B‐1), confirming previous results of Hermo et al. [Anat. Rec. 229:31‐50, 1991]. However, among testicular somatic cells, microtubules of some Sertoli cell branches were not acetylated but glutamylated. 2‐D PAGE of mouse and hamster sperm extracts showed a high level of α and β‐tubulin heterogeneity, comparable to that found in brain. Immunoblotting with GT335 revealed a large amount of glutamylated tubulin resolved into numerous α as well as β‐tubulin isoforms. This suggests that the major testis‐specific tubulin isotypes (mα3/7 and mβ3) are also glutamylatable. These results show a subcellular sorting of posttranslationally modified tubulin isoforms in spermatids, glutamylation being associated with the most stable microtubule structures. © 1994 Wiley‐Liss, Inc.
Role of microtubules in random cell migration: Stabilization of cell polarity
Authors: Glasgow James E. , Daniele Ronald P.
Published: February 2005

The role of microtubules in random cell migration was investigated using time‐lapse videomicroscopy to record in vitro the shape and motile behavior of guinea pig alveolar macrophages before and after disrupting microtubules with colcemid. Cell migration was quantified in terms of directional persistence time and speed. Motility was also correlated with morphological polarity: cells having a single lamellipodal region (monopolar cells) migrated, whereas those lacking a lamellipod (apolar cells) or with opposing lamellipodal regions (bipolar cells) did not migrate. Within 2 hours, colcemid caused a shift in polarity from 80% monopolar cells to 40% monopolar and 40% bipolar cells and a corresponding decrease from 80% to 40% in the fraction of migrating cells. Mean persistence time and speed decreased only slightly (approximately 20%) for those cells (still monopolar) which continued to migrate in the presence of colcemid. Persistence time and speed actually increased for many individual cells, indicating that random migration did not require intact microtubules. We conclude that colcemid treatment destabilizes monopolarity, leading to the gradual loss of monopolarity and consequent inhibition of migration. While a cell remains monopolar, it will continue to migrate even in the absence of intact microtubules, but microtubules are required for the long‐term maintenance of cellular monopolarity and, thus, for continued motility. © 1994 Wiley‐Liss, Inc.
Cellular microtubules heterogeneous in their content of microtubule‐associated protein 4 (MAP4)
Authors: Chapin Steven J. , Bulinski Jeannette Chloë
Published: February 2005

Previous immunolocalization studies using many primate cultured cell lines demonstrated that a microtubule‐associated protein of Mr ∼210,000 which is now called MAP4, is present along the length of microtubules in interphase and mitotic cells [Bulinski and Borisy (1980) J. Cell Biol. 87:802–808; DeBrabander et al. (1981) J. Cell Biol. 91:438–455]. Since MAP4 has been implicated as a microtubule stabilizer, we asked whether all classes of microtubules possess an equal complement of MAP4. We have reexamined the cellular distribution of MAP4, using both conventional double‐label immunofluorescence and an antibody blocking technique [Schulze and Kirschner (1987) J. Cell Biol. 104:277–288] to highlight microtubules lacking, or depleted in, MAP4. These techniques have revealed that thin processes extending from monkey kidney cells (TC‐7), and those made by human neuroblastoma cells (IMR‐32) in response to retinoic acid, are often deficient in MAP4 immunoreactivity. Since both types of cellular processes contain stable microtubules, which are enriched in detyrosinated (Glu) tubulin, we tested the ability of MAP4 to bind to microtubules made from pure Glu and pure tyrosinated (Tyr) tubulin in vitro. MAP4 bound to both types of microtubules, and the similar saturation level of MAP4 binding to Glu and Tyr microtubules suggested that differential binding to these forms of tubulin does not contribute directly to a mechanism for segregation of MAP4 on microtubules in vivo. In TC‐7 cells, we also observed MAP4‐depletion on single microtubules, distal regions of broad cytoplasmic extensions, and midbodies of dividing cells. MAP4 depletion may reflect recent, rapid growth of microtubules to which MAP4 has not yet bound, or the presence of other MAPs that may compete with MAP4 for binding sites on the MT. We suggest that different levels of MAP4 on microtubules may directly modulate microtubule dynamics within single cells, as well as other microtubule functions such as those involving microtubule motor activity. © 1994 Wiley‐Liss, Inc.
Axonemes paralyzed by the presence of dyneins unable to use ribose‐modified ATP
Authors: Lark Ellen , Omoto Charlotte K.
Published: February 2005

Substrate analogs are useful for studying the structures of active sites and for distinguishing between similar enzyme activities. Fluorescent ribose‐modified ATP analogs were used to investigate the functional differences between dynein ATPases. These analogs reactivate (support the movement of) sea urchin sperm axonemes, yet they do not reactivate wild‐type Chalmydomonas axonemes. Surprisingly, the analogs reactivate the axonemes of mutants completely missing the outer arm dyneins. Competition experiments using ATP and these analogs provide strong evidence that the analogs bind to all dynein active sites but fail to release a subset of dyneins from rigor. We suggest that this subset of Chlamydomonas outer arm dyneins unable to use the analogs remains in rigor in the presence of the analogs and paralyzes the axoneme. © 1994 Wiley‐Liss, Inc.
Different temporal patterns of expression result in the same type, amount, and distribution of filamin (ABP) in cardiac and skeletal myofibrils
Authors: Price Maureen G. , Caprette David R. , Gomer Richard H.
Published: February 2005

The morphogenesis of functional myofibrils in chick skeletal and cardiac muscle occurs in greatly different time spans, in about 7 and 2 days, respectively. In chick skeletal myogenic cells, one isoform of the 250 kD actin‐binding protein (ABP) filamin is associated with stress fiber‐like structures of myoblasts and early myotubes, then disappears for approximately 4 days, whereupon a second filamin isoform reappears at the Z‐disc periphery. We sought to determine if cardiac myogenesis involves this sequence of appearance, disappearance, and reappearance of a new filamin isoform in a compressed time scale. It was known that in mature heart, filamin is localized at the Z‐disc periphery as in mature (fast) skeletal muscle, and is also associated with intercalated discs. We find that myocardial filamin has an apparent molecular weight similar to that of adult skeletal muscle filamin and lower than that of smooth muscle filamin, and that both skeletal and cardiac muscle contain roughly 200 filamin monomers per sarcomere. Two‐dimensional peptide mapping shows that myocardial filamin is very similar to skeletal muscle filamin. Myocardial, slow skeletal, and fast skeletal muscle filamins are all phosphorylated, as previously shown for filamin of non‐striated muscle. Using immunofluorescence, we found that filamin could not be detected in the developing heart until the 14‐somite stage, when functional myofibrils exist and the heart has been beating for 3 to 4 hours. We conclude that in cardiac and skeletal myogenesis, different sequences of filamin gene expression result in myofibrils with similar filamin distributions and isoforms. © 1994 Wiley‐Liss, Inc.
Physical model of axonemal splitting
Authors: Holwill Michael E. J. , Satir Peter
Published: February 2005

A physical model developed to explain microtubule sliding patterns in the trypsintreated ciliary axoneme has been extended to investigate the generation of bending moments by microtubules sliding in an axoneme in which the dublets are anchored at one end. With sliding restricted, a bending moment is developed by the polarized shearing interaction between neighbouring doublets, effected by the activity of dynein arms on doublet N pushing N + 1 in a tipward ( + ) direction. In arrested axonemes in which arms on several contiguous doublets are active, the bending moment causes splitting of the 9 + 2 microtubule array into two or more sets of doublets. In the absence of special constraints, splitting depends only on breaking the circumferential interdoublet links most distorted by the bending moment. The analysis, which permits assignment of arm activity to specific microtubules in each of the observed patterns of splitting, indicates that the axoneme will split between doublet N and N + 1 if arms on doublet N are inactive and arms on either N + 1 or N−1 are active. To produce the observed major splits, dynein arms on the microtubules of roughly one‐half of the axoneme are predicted to be active, in a manner consistent with the switch‐point hypothesis of ciliary motion. Electron microscopic examination indicates that virtually every set of doublets in the split axonemes retains its cylindrical form. Maintenance of cylindrical symmetry can be ascribed to the mechanical properties of the unbroken links, which may resist both tensile and compressive stress, and to active dynein arms. © 1994 Wiley‐Liss, Inc.
Localization of tau and other proteins of isolated marginal bands
Authors: Sanchez Ivelisse , Cohen William D.
Published: February 2005

To determine which proteins were associated with and intrinsic to the marginal band (MB) of microtubules (MTs), we studied protein components of MBs isolated from nucleated erythrocytes by differential detergent solubilization of the membrane skeleton (MS). MBs isolated from dogfish erythrocytes contained major proteins in the tubulin Mr range. A high molecular weight protein of ∼290 kD that bound antibody to syncolin and to heat‐stable brain MAPs was present in the whole cytoskeleton. However, most of it was solubilized by the MB isolation medium, together with the MS. Dogfish erythrocyte cytoskeletons and isolated MBs were examined with polyclonal and monoclonal antibodies against mammalian brain tau and chicken erythrocyte tau. As shown by immunofluorescence and immunoblotting, these antibodies bound to proteins in the 50 to 67 kD range, located along the length of isolated MBs. Two‐dimensional SDS‐PAGE revealed isolated MB proteins of pI ∼6.8 in the same molecular weight range, as well as α‐ and β‐tubulin with pI ∼5.4. Subtilisin or high‐salt treatment of isolated MBs resulted in unbundling of MTs, indicating involvement of MAPs. MBs isolated from chicken erythrocyte cytoskeletons also contained tau as shown by antimammalian brain tau immunofluorescence. Both chicken and dogfish isolated MBs also bound phalloidin, but the binding was usually discontinuous and, for any given MB, matched the pattern of anti‐syncolin binding. Both syncolin and F‐actin were part of the MS remnant remaining after MT disassembly, supporting their assignment to a specialized MS region at the MB/MS interface. In contrast, tau protein appears to be intrinsic to the MB, where it may have an MT stabilizing and bundling function. © 1994 Wiley‐Liss, Inc.
Actin cytoskeleton and motility in rat sarcoma cell populations with different metastatic potential
Authors: Pokorná E. , Jordan P. W. , O'Neill C. H. , Zicha D. , Gilbert C. S. , Veselý P.
Published: February 2005

We have studied the organisation of the actin cytoskeleton in three related rat sarcoma cell populations of differing malignancy. They were derived by neoplastic progression from a population which had transformed spontaneously in vitro, and were distinguished by their ability to give rise to reproducibly different numbers of metastases, ranging from 10% to 80% of the animals inoculated. We found characteristic differences in the arrangement of the actin cytoskeleton. Confocal three‐dimensional microscopy showed that nearly all of the least malignant population contained conspicuous actin stress fibres lying in the lower part of the cell parallel to the substratum and no other actin structures. Actin in the intermediate population was typically situated in a diffuse layer underlying the whole plasma membrane, in which no fibres could be seen. Two thirds of the most malignant population consisted of more rounded cells filled with a three‐dimensional network of fine oblique actin fibres. There were focal contacts in all these cells; their area showed a regular decrease from 1.3 μm2 to 0.4 μm2. The differences in actin distribution were accompanied by differences in motility, which increased as malignancy increased. When individual cells were fixed after they had been tracked by time‐lapse, their cytoskeleton type correlated with the speed at which they had moved. All these differences were enhanced at low pH. These findings point to the possibility that the three‐dimensional network of fine actin fibres in acid culture could be a measure of the malignant potential of transformed cells in vitro. © 1994 Wiley‐Liss, Inc.
Are microtubules essential for the secretory process in rat parotid gland?
Authors: Robin Philippe , Rossignol Bernard , Raymond Marie‐Noëlle
Published: February 2005

The role of microtubules in the exocrine secretory process is not yet well established, and their disruption by anti‐microtubule drugs leads to variable effects on intracellular transit and protein secretion. We investigated the involvement of microtubules in the regulated secretory process of rat parotid glands using microscopic techniques and pulse‐chase experiments. We showed that 10 μM colchicine or nocodazole destroys the microtubule network in parotid acinar cells but only weakly reduces the release of newly synthesized proteins. The half‐effect was obtained with 0.22 μM colchicine. Moreover, this small reduction was found to be independent of the nature of the drug (colchicine, colcemid, or nocodazole) and of the nature of the stimulation (β‐adrenergic or cholinergic pathways). Using nocodazole, we have been able to determine that the steps affected by the drug are very early events in the secretory pathway. Finally, we showed by kinetic analysis that microtubule disruption slows protein release only moderately but does not reduce the total amount of secreted protein. We conclude from this study that microtubule integrity is not essential for protein secretion in rat parotid gland. © 1994 Wiley‐Liss, Inc.
Microtubule‐associated protein function: Lessons from expression in spodoptera frugiperda cells
Authors: Kosik Kenneth S. , McConlogue Lisa
Published: February 2005

The phenotypes induced by the expression of neuronal microtubule‐associated proteins (MAPs) in Sf9 cells have provided data on the in situ function of these proteins. Both MAP2 and tau can induce long processes in Sf9 cells, and the processes contain bundles of microtubules. In both cases the microtubules are aligned with their plus ends distal. Tau expression usually induces a single process that is unbranched and of uniform caliber. Processes can form even when the cells are grown in suspension. Microtubules do not extend all the way to the tip; instead the terminal region contains an actin‐rich meshwork. Taxol treatment of Sf9 cells also induces the assembly of microtubules into bundles but does not induce process formation in Sf9 cells. Therefore the in vitro properties of tau as a molecule capable of assembling, stabilizing, and bundling microtubules do not fully account for the in vivo ability of tau alone to transduce microtubule assembly into a change in cell shape. The morphological features of the processes induced by MAP2 differ in highly informative ways. © 1994 Wiley‐Liss, Inc.
Organization of actin and microtubules during process formation in tau‐expressing sf9 cells
Authors: Knowles Roger , Leclerc Nicole , Kosik Kenneth S.
Published: February 2005

Insect Sf9 cells usually elaborate a highly characteristic single process when infected with a baculovirus encoding recombinant human tau. The processes are unbranched, of uniform caliber, and contain bundles of microtubules. Because taxol treatment alone does not induce process outgrowth in these cells, it is believed that tau confers properties on microtubules that permits the conversion of microtubule assembly into the formation of processes. Here we have analyzed the reorganization of both actin filaments and microtubules during process initiation. A zone of organelle exclusion representing the focal reorganization of actin at one pole of the cell anticipated process emergence. A relationship between actin organization and process emergence was also suggested by a shift from single to multiple process formation after treatment with cytochalasin D. The rate of process elongation doubled after cytochalasin treatment of tau‐expressing cells. The increase in rate was due to the inhibition of the growth arrest phases which occur in the absence of cytochalasin. In contrast, Sf9 cells treated with cytochalasin after more than 20 h of tau expression were relatively resistant to the drug's effects. We conclude that actin and microtubules are specifically reorganized during tau‐induced process outgrowth and that a dynamic relationship between actin filaments and microtubules effects process formation. © 1994 Wiley‐Liss, Inc.
Rhazinilam mimics the cellular effects of taxol by different mechanisms of action
Authors: David Bruno , Sévenet Thierry , Morgat Michel , Guénard Daniel , Moisand André , Tollon Yvette , Thoison Odile , Wright Michel
Published: February 2005

We have investigated the effects of the microtubule poison rhazinilam on microtubule assembly in vivo and in vitro. In mammalian cells, rhazinilam mimics the effects of taxol and leads to microtubule bundles, multiple asters, and microtubule cold stability. In vitro, rhazinilam protected preassembled microtubules from cold‐induced disassembly, but not from calcium ion‐induced disassembly. Moreover, both at 0°C and at 37°C, rhazinilam induced the formation of anomalous tubulin assemblies (spirals). This process was prevented by maytansine and vinblastine, but not by colchicine. Preferential saturable and stoichiometric binding of radioactive rhazinilam to tubulin in spirals was observed with a dissociation constant of 5 μM. This binding was abolished in the presence of vinblastine and maytansine. In contrast, specific binding of radioactive rhazinilam to tubulin assembled in microtubules was undetectable. These results demonstrate that rhazinilam alters microtubule stability differently than taxol, and that the overall similar effects of rhazinilam and taxol on the cellular cytoskeleton are the consequence of two distinct mechanisms of action at the molecular level. © 1994 Wiley‐Liss, Inc.
Dynamics of actin and alpha‐actinin in the tails of Listeria monocytogenes in Infected PtK2 Cells
Authors: Nanavati Dipali , Ashton Francis T. , Sanger Jean M. , Sanger Joseph W.
Published: February 2005

Listeria monocytogenes can penetrate and multiply within a variety of cell types, including the PtK2 kidney epithelial line. Once released within the cytoplasm, L. monocytogenes acquires the capacity for rapid movement through the host cell [Dabiri et al., 1990: Proc. Natl. Acad. Sci. 87:6068‐6072]. In the process, actin monomers are inserted in proximity to one end of the bacterium, forming a column or tail of actin filaments [Sanger et al., 1992: Infect. Immun. 60:3609‐3619]. The rate of new actin filament growth correlates closely with the speed of bacterial migration. In this study we have used fluorescently labeled actin and alpha‐actinin to monitor the movement and turnover rate of actin and alpha‐actinin molecules in the tails. The half‐lives of the actin and alpha‐actinin present in the tails are approximately the same: actin, 58.7 sec; alpha‐actinin, 55.3 sec. The half‐life of alpha‐actinin surrounding a dividing bacterium was 30 sec, whereas its half‐life in the tails that formed behind the two daughter cells was about 20‐30% longer. We discovered that the speeds of the bacteria are not constant, but show aperiodic episodes of decreased and increased speeds. There is a fluctuation also in the intensities of the fluorescent probes at the bacterium/tail interface, implying that there is a fluctuation in the number of actin filaments forming there. There was no strong correlation, however, between these fluctuating intensities and changes in speed of the bacteria. These measurements suggest that while actin polymerization at the bacterial surface is coupled to the movement of the bacterium, the periodic changes in intracellular motility are not a simple function of the number of actin filaments nucleating at the bacterial surfaces. © 1994 Wiley‐Liss, Inc.