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PubMed1983

Occluded and Budded Autographa californica Nuclear Polyhedrosis Virus: Immunological Relatedness of Structural Proteins.

The immunological relatedness of the structural proteins of the budded and occluded phenotypes of Autographa californica nuclear polyhedrosis virus was examined by reciprocal immunoblotting and by in situ peroxidase-antiperoxidase staining of virus-induced cell surface and intracellular antigens with antisera to both phenotypes. The molecular weights (MWs) of major structural proteins of both phenotypes that reciprocally cross-reacted were 92,500, 78,000, 62,500, 54,000, and 42,000. A highly immunogenic, major structural protein of the occluded phenotype of 46,000 MW was not recognized by antiserum to the budded phenotype, and a major structural protein of the budded phenotype, 48,000 MW, was not recognized by antiserum to the occluded phenotype. Both the budded and occluded phenotypes contained a protein of 33,500 MW that comigrated with polyhedrin (the matrix protein) and reacted with antiserum and monoclonal antibody to polyhedrin. Evidence was obtained for the apparent antigenic relatedness of proteins of different MWs from the budded and occluded phenotypes, possibly indicative of different processing of some proteins for the two phenotypes. Antiserum to the occluded phenotype recognized virus-induced cell surface antigens, indicating antigenic similarities between the occluded phenotype and envelope proteins of the budded phenotype. Antiserum to the budded phenotype recognized viral proteins produced before the appearance of cytopathic effect, whereas antiserum to the occluded phenotype did not.

Journal Article↗

PubMed1994

Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding.

The prevailing hypothesis is that the intracellular site of budding of coronaviruses is determined by the localization of its membrane protein M (previously called E1). We tested this by analyzing the site of budding of four different coronaviruses in relation to the intracellular localization of their M proteins. Mouse hepatitis virus (MHV) and infectious bronchitis virus (IBV) grown in Sac(-) cells, and feline infectious peritonitis virus (FIPV) and transmissible gastroenteritis virus (TGEV) grown in CrFK cells, all budded exclusively into smooth-walled, tubulovesicular membranes located intermediately between the rough endoplasmic reticulum and Golgi complex, identical to the so-called budding compartment previously identified for MHV. Indirect immunofluorescence staining of the infected cells showed that all four M proteins accumulated in a perinuclear region. Immunogold microscopy localized MHV M and IBV M in the budding compartment; in addition, a dense labeling in the Golgi complex occurred, MHV M predominantly in trans-Golgi cisternae and trans-Golgi reticulum and IBV M mainly in the cis and medial Golgi cisternae. The corresponding M proteins of the four viruses, when independently expressed in a recombinant vaccinia virus system, also accumulated in the perinuclear area. Quantitative pulse-chase analysis of metabolically labeled cells showed that in each case the majority of the M glycoproteins carried oligosaccharide side chains with Golgi-specific modifications within 4 h after synthesis. Immunoelectron microscopy localized recombinant MHV M and IBV M to the same membranes as the respective proteins in coronavirus-infected cells, with the same cis-trans distribution over the Golgi complex. Our results demonstrate that some of the M proteins of the four viruses are transported beyond the budding compartment and are differentially retained by intrinsic retention signals; in addition to M, other viral and/or cellular factors are probably required to determine the site of budding.

Animals↗

PubMed2000

Semliki forest virus budding: assay, mechanisms, and cholesterol requirement.

All enveloped viruses must bud through a cellular membrane in order to acquire their lipid bilayer, but little is known about this important stage in virus biogenesis. We have developed a quantitative biochemical assay to monitor the budding of Semliki Forest virus (SFV), an enveloped alphavirus that buds from the plasma membrane in a reaction requiring both viral spike proteins and nucleocapsid. The assay was based on cell surface biotinylation of newly synthesized virus spike proteins and retrieval of biotinylated virions using streptavidin-conjugated magnetic particles. Budding of biotin-tagged SFV was continuous for at least 2 h, independent of microfilaments and microtubules, strongly temperature dependent, and relatively independent of continued exocytic transport. Studies of cell surface spike proteins at early times of infection showed that these spikes did not efficiently bud into virus particles and were rapidly degraded. In contrast, at later times of infection, spike protein degradation was markedly reduced and efficient budding was then observed. The previously described cholesterol requirement in SFV exit was shown to be due to a block in budding in the absence of cholesterol and correlated with the continued degradation of spike proteins at all times of virus infection in sterol-deficient cells.

Animals↗

PubMed2001

Role of matrix and fusion proteins in budding of Sendai virus.

Paramyxoviruses are assembled at the surface of infected cells, where virions are formed by the process of budding. We investigated the roles of three Sendai virus (SV) membrane proteins in the production of virus-like particles. Expression of matrix (M) proteins from cDNA induced the budding and release of virus-like particles that contained M, as was previously observed with human parainfluenza virus type 1 (hPIV1). Expression of SV fusion (F) glycoprotein from cDNA caused the release of virus-like particles bearing surface F, although their release was less efficient than that of particles bearing M protein. Cells that expressed only hemagglutinin-neuraminidase (HN) released no HN-containing vesicles. Coexpression of M and F proteins enhanced the release of F protein by a factor greater than 4. The virus-like particles containing F and M were found in different density gradient fractions of the media of cells that coexpressed M and F, a finding that suggests that the two proteins formed separate vesicles and did not interact directly. Vesicles released by M or F proteins also contained cellular actin; therefore, actin may be involved in the budding process induced by viral M or F proteins. Deletion of C-terminal residues of M protein, which has a sequence similar to that of an actin-binding domain, significantly reduced release of the particles into medium. Site-directed mutagenesis of the cytoplasmic tail of F revealed two regions that affect the efficiency of budding: one domain comprising five consecutive amino acids conserved in SV and hPIV1 and one domain that is similar to the actin-binding domain required for budding induced by M protein. Our results indicate that both M and F proteins are able to drive the budding of SV and propose the possible role of actin in the budding process.

Actins↗

PubMed2002

Roles for the cytoplasmic tails of the fusion and hemagglutinin-neuraminidase proteins in budding of the paramyxovirus simian virus 5.

The efficient release of many enveloped viruses from cells involves the coalescence of viral components at sites of budding on the plasma membrane of infected cells. This coalescence is believed to require interactions between the cytoplasmic tails of surface glycoproteins and the matrix (M) protein. For the paramyxovirus simian virus 5 (SV5), the cytoplasmic tail of the hemagglutinin-neuraminidase (HN) protein has been shown previously to be important for normal virus budding. To investigate a role for the cytoplasmic tail of the fusion (F) protein in virus assembly and budding, we generated a series of F cytoplasmic tail-truncated recombinant viruses. Analysis of these viruses in tissue culture indicated that the cytoplasmic tail of the F protein was dispensable for normal virus replication and budding. To investigate further the requirements for assembly and budding of SV5, we generated two double-mutant recombinant viruses that lack 8 amino acids of the predicted 17-amino-acid HN protein cytoplasmic tail in combination with truncation of either 10 or 18 amino acids from the predicted 20-amino-acid F protein cytoplasmic tail. Both of the double mutant recombinant viruses displayed a replication defect in tissue culture and a budding defect, the extent of which was dependent on the length of the remaining F cytoplasmic tail. Taken together, this work and our earlier data on virus-like particle formation (A. P. Schmitt, G. P. Leser, D. L. Waning, and R. A. Lamb, J. Virol. 76:3953-3964, 2002) suggest a redundant role for the cytoplasmic tails of the HN and F proteins in virus assembly and budding.

Amino Acid Sequence↗

PubMed2003

Defects in human immunodeficiency virus budding and endosomal sorting induced by TSG101 overexpression.

Retrovirus budding is greatly stimulated by the presence of Gag sequences known as late or L domains. The L domain of human immunodeficiency virus type 1 (HIV-1) maps to a highly conserved Pro-Thr-Ala-Pro (PTAP) sequence in the p6 domain of Gag. We and others recently observed that the p6 PTAP motif interacts with the cellular endosomal sorting protein TSG101. Consistent with a role for TSG101 in virus release, we demonstrated that overexpressing the N-terminal, Gag-binding domain of TSG101 (TSG-5') suppresses HIV-1 budding by blocking L domain function. To elucidate the role of TSG101 in HIV-1 budding, we evaluated the significance of the binding between Gag and TSG-5' on the inhibition of HIV-1 release. We observed that a mutation in TSG-5' that disrupts the Gag/TSG101 interaction suppresses the ability of TSG-5' to inhibit HIV-1 release. We also determined the effect of overexpressing a panel of truncated TSG101 derivatives and full-length TSG101 (TSG-F) on virus budding. Overexpressing TSG-F inhibits HIV-1 budding; however, the effect of TSG-F on virus release does not require Gag binding. Furthermore, overexpression of the C-terminal portion of TSG101 (TSG-3') potently inhibits budding of not only HIV-1 but also murine leukemia virus. Confocal microscopy data indicate that TSG-F and TSG-3' overexpression induces an aberrant endosome phenotype; this defect is dependent upon the C-terminal, Vps-28-binding domain of TSG101. We propose that TSG-5' suppresses HIV-1 release by binding PTAP and blocking HIV-1 L domain function, whereas overexpressing TSG-F or TSG-3' globally inhibits virus release by disrupting the cellular endosomal sorting machinery. These results highlight the importance of TSG101 and the endosomal sorting pathway in virus budding and suggest that inhibitors can be developed that, like TSG-5', target HIV-1 without disrupting endosomal sorting.

Amino Acid Motifs↗

PubMed2003

Plasma membrane budding as an alternative release mechanism of the extracellular enveloped form of vaccinia virus from HeLa cells.

In HeLa cells the assembly of modified vaccinia virus Ankara (MVA), an attenuated vaccinia virus (VV) strain, is blocked. No intracellular mature viruses (IMVs) are made and instead, immature viruses accumulate, some of which undergo condensation and are released from the cell. The condensed particles may undergo wrapping by membranes of the trans-Golgi network and fusion with the plasma membrane prior to their release (M. W. Carroll and B. Moss, Virology 238:198-211, 1997). The present study shows by electron microscopy (EM), however, that the dense particles made in HeLa cells are also released by a budding process at the plasma membrane. By labeling the plasma membrane with antibodies to B5R, a membrane protein of the extracellular enveloped virus, we show that budding occurs at sites that concentrate this protein. EM quantitation revealed that the cell surface around a budding profile was as strongly labeled with anti-B5R antibody as were the extracellular particles, whereas the remainder of the plasma membrane was significantly less labeled. To test whether budding was a characteristic of MVA infection, HeLa cells were infected with the replication competent VV strains Western Reserve strain (WR) and International Health Department strain-J (IHD-J) and also prepared for EM. EM analyses, surprisingly, revealed for both virus strains IMVs that evidently budded at the cell surface at sites that were significantly labeled with anti-B5R. EM also indicated that budding of MVA dense particles was more efficient than budding of IMVs from WR- or IHD-J-infected cells. This was confirmed by semipurifying [(35)S]methionine-labeled dense particles or extracellular enveloped virus (EEVs) from the culture supernatant of MVA- or IHD-J-infected HeLa cells, respectively, showing that threefold more labeled dense particles were secreted than EEVs. Finally, although the released MVA dense particles contain some DNA, they are not infectious, as assessed by plaque assays.

Animals↗

PubMed2003

Retroviruses have differing requirements for proteasome function in the budding process.

Proteasome inhibitors reduce the budding of human immunodeficiency virus types 1 (HIV-1) and 2, simian immunodeficiency virus, and Rous sarcoma virus. To investigate this effect further, we examined the budding of other retroviruses from proteasome inhibitor-treated cells. The viruses tested differed in their Gag organization, late (L) domain usage, or assembly site from those previously examined. We found that proteasome inhibition decreased the budding of murine leukemia virus (plasma membrane assembly, PPPY L domain) and Mason-Pfizer monkey virus (cytoplasmic assembly, PPPY L domain), similar to the reduction observed for HIV-1. Thus, proteasome inhibitors can affect the budding of a virus that assembles within the cytoplasm. However, the budding of mouse mammary tumor virus (MMTV; cytoplasmic assembly, unknown L domain) was unaffected by proteasome inhibitors, similar to the proteasome-independent budding previously observed for equine infectious anemia virus (plasma membrane assembly, YPDL L domain). Examination of MMTV particles detected Gag-ubiquitin conjugates, demonstrating that an interaction with the ubiquitination system occurs during assembly, as previously found for other retroviruses. For all of the cell lines tested, the inhibitor treatment effectively inactivated proteasomes, as measured by the accumulation of polyubiquitinated proteins. The ubiquitination system was also inhibited, as evidenced by the loss of monoubiquitinated histones from treated cells. These results and those from other viruses show that proteasome inhibitors reduce the budding of viruses that utilize either a PPPY- or PTAP-based L domain and that this effect does not depend on the assembly site or the presence of monoubiquitinated Gag in the virion.

Animals↗

PubMed2003

APC mutation and tumour budding in colorectal cancer.

AIM: To determine the frequency of tumour budding and somatic APC mutation in a series of colorectal cancers stratified according to DNA microsatellite instability (MSI) status. MATERIAL/METHODS: Ninety five colorectal cancers were genotyped for APC mutation in the mutation cluster region (exon 15) and scored for the presence of tumour budding at the invasive margin in haematoxylin and eosin stained sections. A subset was immunostained for beta catenin and p16. RESULTS: The frequency of both somatic APC mutation and tumour budding increased pari passu in cancers stratified as sporadic MSI high (MSI-H), hereditary non-polyposis colorectal cancer (HNPCC), MSI low (MSI-L), and microsatellite stable (MSS). Both budding and APC mutation were significantly less frequent in sporadic MSI-H cancers than in MSI-L or MSS cancers. Tumour buds were characterised by increased immunostaining for both beta catenin and p16. CONCLUSION: Tumour budding is associated with an adverse prognosis. The lack of budding in MSI-H colorectal cancer may account for the improved prognosis of this subset and may be explained by an intact WNT signalling pathway and/or inactivated p16(INK4a).

Biomarkers, Tumor↗

PubMed1990

Gradients of homeoproteins in developing feather buds.

Homeoproteins are functionally involved in pattern formation. Recently, homeoproteins have been shown to be distributed in a graded fashion in developing limb buds. Here we examine the expression of homeoproteins in chicken feather development by immunocytochemical localization. We find that XlHbox 1 antigen is present in cell nuclei and is distributed in a gradient in the mesoderm of developing feather buds, with strongest expression in the anterior-proximal region. The gradient is most obvious in feather buds from the mid-trunk level. Feather buds from the scapular level express very high levels of XlHbox 1 and feather buds from the caudal region express no XlHbox 1, suggesting that a broad gradient along the body axis is superimposed on a smaller gradient within each individual feather bud. Feather ectoderm also expresses XlHbox 1 antigen but without an obvious graded pattern. Another homeoprotein, Hox 5.2, is also expressed in developing feather buds in a graded way, and its distribution pattern is partially complementary to that of XlHbox 1. These observations suggest that homeoproteins may be involved in setting up the anteroposterior polarity of cell fields at different levels, first for the body axis, then for the limb axis and finally for the feather axis.

Animals↗

PubMed1995

Formin isoforms are differentially expressed in the mouse embryo and are required for normal expression of fgf-4 and shh in the limb bud.

Mice homozygous for the recessive limb deformity (ld) mutation display both limb and renal defects. The limb defects, oligodactyly and syndactyly, have been traced to improper differentiation of the apical ectodermal ridge (AER) and shortening of the anteroposterior limb axis. The renal defects, usually aplasia, are thought to result from failure of ureteric bud outgrowth. Since the ld locus gives rise to multiple RNA isoforms encoding several different proteins (termed formins), we wished to understand their role in the formation of these organs. Therefore, we first examined the embryonic expression patterns of the four major ld mRNA isoforms. Isoforms I, II and III (all containing a basic amino terminus) are expressed in dorsal root ganglia, cranial ganglia and the developing kidney including the ureteric bud. Isoform IV (containing an acidic amino terminus) is expressed in the notochord, the somites, the apical ectodermal ridge (AER) of the limb bud and the developing kidney including the ureteric bud. Using a lacZ reporter assay in transgenic mice, we show that this differential expression of isoform IV results from distinct regulatory sequences upstream of its first exon. These expression patterns suggest that all four isoforms may be involved in ureteric bud outgrowth, while isoform IV may be involved in AER differentiation. To define further the developmental consequences of the ld limb defect, we analyzed the expression of a number of genes thought to play a role in limb development. Most significantly, we find that although the AERs of ld limb buds express several AER markers, they do not express detectable levels of fibroblast growth factor 4 (fgf-4), which has been proposed to be the AER signal to the mesoderm. Thus we conclude that one or more formins are necessary to initiate and/or maintain fgf-4 production in the distal limb. Since ld limbs form distal structures such as digits, we further conclude that while fgf-4 is capable of supporting distal limb outgrowth in manipulated limbs, it is not essential for distal outgrowth in normal limb development. In addition, ld limbs show a severe decrease in the expression of several mesodermal markers, including sonic hedgehog (shh), a marker for the polarizing region and Hoxd-12, a marker for posterior mesoderm. We propose that incomplete differentiation of the AER in ld limb buds leads to reduction of polarizing activity and defects along the anteroposterior axis.

Animals↗

PubMed1996

IGF-I, insulin and FGFs induce outgrowth of the limb buds of amelic mutant chick embryos.

IGF-I, insulin, FGF-2 and FGF-4 have been implicated in the reciprocal interactions between the apical ectodermal ridge (AER) and underlying mesoderm required for outgrowth and patterning of the developing limb. To study further the roles of these growth factors in limb outgrowth, we have examined their effects on the in vitro morphogenesis of limb buds of the amelic mutant chick embryos wingless (wl) and limbless (ll). Limb buds of wl and ll mutant embryos form at the proper time in development, but fail to undergo further outgrowth and subsequently degenerate. Wl and ll limb buds lack thickened AERs capable of promoting limb outgrowth, and their thin apical ectoderms fail to express the homeobox-containing gene Msx-2, which is highly expressed by normal AERs and has been implicated in regulating AER activity. Here we report that exogenous IGF-I and insulin, and, to a lesser extent, FGF-2 and FGF-4 induce the proliferation and directed outgrowth of explanted wl and ll mutant limb buds, which in vitro, like in vivo, normally fail to undergo outgrowth and degenerate. IGF-I and insulin, but not FGFs, also cause the thin apical ectoderms of wl and ll limb buds to thicken and form structures that grossly resemble normal AERs and, moreover, induce high level expression of Msx-2 in these thickened AER-like structures. Neither IGF-I, insulin nor FGFs induce expression of the homeobox-containing gene Msx-1 in the subapical mesoderm of wl or ll limb buds, although FGFs, but not IGF-I or insulin, maintain Msx-1 expression in normal (non-mutant) limb bud explants lacking an AER. The implications of these results to the relationships among the wl and ll genes, IGF-I/insulin, FGFs, Msx-2 and Msx-1 in the regulation of limb outgrowth is discussed.

Animals↗

PubMed2003

COPII proteins are required for Golgi fusion but not for endoplasmic reticulum budding of the pre-chylomicron transport vesicle.

The budding of vesicles from endoplasmic reticulum (ER) that contains nascent proteins is regulated by COPII proteins. The mechanisms that regulate lipid-carrying pre-chylomicron transport vesicles (PCTVs) budding from the ER are unknown. To study the dependence of PCTV-ER budding on COPII proteins we examined protein and PCTV budding by using ER prepared from rat small intestinal mucosal cells prelabeled with (3)H-oleate or (14)C-oleate and (3)H-leucine. Budded (3)H-oleate-containing PCTVs were separated by sucrose density centrifugation and were revealed by electron microscopy as 142-500 nm vesicles. Our results showed the following: (1) Proteinase K treatment did not degrade the PCTV cargo protein, apolipoprotein B-48, unless Triton X-100 was added. (2) PCTV budding was dependent on cytosol and ATP. (3) The COPII proteins Sar1, Sec24 and Sec13/31 and the membrane proteins syntaxin 5 and rBet1 were associated with PCTVs. (4) Isolated PCTVs were able to fuse with intestinal Golgi. (5) Antibodies to Sar1 completely inhibited protein vesicle budding but increased the generation of PCTV; these changes were reversed by the addition of recombinant Sar1. (6) PCTVs formed in the absence of Sar1 did not contain the COPII proteins Sar1, Sec24 or Sec31 and did not fuse with the Golgi complex. Together, these findings suggest that COPII proteins may not be required for the exit of membrane-bound chylomicrons from the ER but that they or other proteins may be necessary for PCTV fusion with the Golgi.

Animals↗

PubMed2005

Male-fertility genes expressed in male flower buds of Silene latifolia include homologs of anther-specific genes.

When the female plant of Silene latifolia is infected with the smut fungus Microbotryum violaceum, its rudimentary stamens develop into anthers which contain fungus teliospores instead of pollen. To identify genes required for maturation of anthers in S. latifolia, we performed a cDNA subtraction approach with healthy male buds and female buds infected with M. violaceum. We isolated five cDNA clones, which were preferentially expressed in healthy male buds during stages associated with a burst in tapetal activity. These five cDNAs are predicted to encode a mandelonitrile lyase protein (SlMDL1), a strictosidine synthase protein (SlSs), a glycosyl hydrolase 17 protein (SlGh17), a proline-rich protein APG precursor (SlAPG), and a chalcone-synthase-like protein (SlChs). All five genes showed expression in both healthy and fungus-infected male buds, but not expressed in either healthy or infected female buds. The first three genes were highly expressed in both tapetum and pollen grains while the last two genes were expressed only inside the tapetum of male flower buds. Phylogenetic analysis results showed that SlChs and SlGh17 belong to anther-specific subgroups of chalcone-synthase-like genes and glycosyl hydrolase 17 family genes, respectively. Our results suggest that the isolated five genes are related to the fertility of the anther leading to the development of fertile pollen. The smut fungus was not able to induce the expression of the five genes in the infected female buds. This raises the possibility that these genes are under the control of master gene(s) on the Y chromosome.

Amino Acid Sequence↗

PubMed2005

[HIV budding and Tsg101].

HIV, as well as many enveloped viruses, exits the cells by budding directly from the plasma membrane. HIV budding is dependent on a PTAP motif, which is located within the p6 domain of Gag. Recent studies have shown that the cellular protein Tsg101 binds to the PTAP L-domain motif of HIV p6 and facilitates the final stages of virus release. Tsg101 function in the cellular vacuolar protein sorting pathway, where they play central roles in selecting cargo for incorporation into vesicles that bud into the maturing endosome to create multivesicular bodies (MVBs). Vesicle budding into the MVB and viral budding at the plasma membrane are topologically equivalent, and the same machinery could catalyze both processes. It will be important to understand the mechanism of virus budding in detail, since virus budding may be a potential target for interference with HIV propagation.

Amino Acid Motifs↗

PubMed2000

[Genistein represses the induction of prostatic buds by testosterone] .

Genistein, a phytoestrogen and a kind of endocrine disrupters, inhibits tyrosine-specific protein kinase activity of the epidermal growth factor (EGF) receptor. It is also effective both in the suppression of the prostatic cell proliferation and the prostate carcinogenesis. We have recently demonstrated that several growth factors, like EGF, transforming growth factor-alpha (TGF-alpha), or keratinocyte growth factor (KGF), can induce prostatic bud formation in the absence of androgen. The present study was performed to investigate whether genistein can suppress testosterone-induced prostatic bud formation. Urogenital sinuses of 16.5-day male rat fetuses were cultured organotypically for 5 days in a serum-free medium containing 10 or 100 ng/ml genistein and 50 ng/ml testosterone. The number and total volume of prostatic buds were analyzed by laser scanning microscopy and computerized. We found that genistein inhibits significantly testosterone-induced prostatic bud formation. In the presence of genistein, cell proliferation of the sinus epithelium was suppressed and the number of prostatic buds and total volume of the buds were reduced as compared with those in the sinuses cultured with testosterone alone. Genistein did not appear to cause necrosis of the sinus. These results support our hypothesis that growth factors like EGF secreted from the sinus mesenchyme activated by testosterone are involved in the induction and stimulation of growth of the prostatic buds.

Androgen Antagonists↗

PubMed2001

Characterization of the CDC10 product and the timing of events of the budding site of Saccharomyces cerevisiae.

Budding cells of the yeast Saccharomyces cerevisiae possess a ring of septin filaments of unknown biochemical nature that lies under the inner surface of the plasma membrane in the neck that connects the mother cell to its bud. Mutants, defective in any of the four genes (CDC3, CDC10, CDC11, CDC12), lack these septin filaments and display a pleiotropic phenotype that involves abnormal bud growth and an inability to complete cytokinesis. The cloned CDC10 was fused to bacterial genes to generate antibodies specific for the CDC10 product was a constituent of the septin filaments. Cdc10p-specific antibodies for septin staining and actin-specific rhodamine-phalloidine were used to investigate the timing of the localization of septin and actin at the budding site using the immunofluorescence microscopic technique. In wild-type cells, the timing of the appearance and disappearance of these proteins was indistinguishable. In addition, the cdc10 mutant did not prevent actin localization at the budding site. The mutant that was blocked in the actin function also did not prevent the septin localization of the Cdc10p. This result may suggest an organizational independence between these proteins in the bud formation. Finally, the localization of septin and actin in the cdc24 mutant cell was examined. It was found that the CDC24 function was necessary for the organization of septin and actin at the budding site.

Actins↗

PubMed2002

Differential cell affinity and sorting of anterior and posterior cells during outgrowth of recombinant avian limb buds.

To examine the role of position-specific differences in cell-cell affinity, recombinant limb buds composed of dissociated and reaggregated cells derived from anterior (A) and posterior (P) limb bud fragments were analyzed. Dissociated anterior and/or posterior cells were differentially labeled, and their behavior was analyzed during recombinant limb bud outgrowth. We find that anterior and posterior cells sort out from one another to form alternating anterior and posterior stripes of cells that extend distally along the proximal-distal axis. These alternating stripes are prominent across the A/P axis in whole-mount preparations of recombinant limb buds after 48 h of outgrowth when the presumptive autopod is dorsal-ventrally flattened and digit rudiments are not evident. After 96 h, when digital and interdigital regions are clearly defined, we find evidence that A/P stripes do not follow obvious anatomical boundaries. The formation of A/P stripes is not inhibited by grafts of ZPA tissue, suggesting that polarizing activity does not influence cell-cell affinity early in limb outgrowth. In vitro studies provide evidence that cell sorting is not dependent on the limb bud ectoderm or the AER; however, cells sort out without organizing into stripes. Gene expression studies using anterior-specific (Alx-4) and posterior-specific (Shh, Bmp-2, and Hoxd-13) marker genes failed to reveal expression domains that corresponded to stripe formation. Control recombinant limb buds composed of anterior, central, or posterior mesenchyme formed digits in a position-specific manner. A/P recombinant limb buds that develop to later stages form digits that are characteristic of central recombinant limbs. These data provide the first definitive evidence of A/P cell sorting during limb outgrowth in vivo and suggest that differential cell affinities play a role in modulating cell behavior during distal outgrowth.

Animals↗