Fascin Homolog Research Articles

Circular RNA circSATB2 promotes progression of non-small cell lung cancer cells

BackgroundLung cancer has high morbidity and mortality worldwide with non-small cell lung cancer (NSCLC) accounting for 85% of the cases. Therapies for lung cancer have relatively poor outcomes and further improvements are required. Circular RNAs have been reported to participate in the occurrence and progression of cancer. Information on the functions and mechanism of circRNAs in lung cancer is limited and needs more exploration.MethodsWe detected expression of genes and proteins by qPCR and western blot. Function of circSATB2 was investigated using RNA interference and overexpression assays. Location of circSATB2 was assessed by fluorescence in situ hybridization (FISH). Interaction of circSATB2, miR-326 and FSCN1 was confirmed by dual-luciferase reporter assay.ResultsData from the investigation showed that circSATB2 was highly expressed in NSCLC cells and tissues. circSATB2 positively regulated fascin homolog 1, actin-bundling protein 1 (FSCN1) expression via miR-326 in lung cancer cells. Furthermore, circSATB2 can be transferred by exosomes and promote the proliferation, migration and invasion of NSCLC cells, as well as induce abnormal proliferation in normal human bronchial epithelial cells. Also, circSATB2 was highly expressed in serumal exosomes from lung cancer patients with high sensitivity and specificity for clinical detection and was related to lung cancer metastasis.ConclusionscircSATB2 participated in the progression of NSCLC and was differentially expressed in lung cancer tissue and serumal exosomes. circSATB2 may be potential biomarker for the diagnosis of NSCLC.

A role for fascin in preventing filopodia breakage in Drosophila tracheal cells

Filopodia are long and thin finger-like protrusions essential for cell migration. They are formed by parallel actin bundles tightly packed by cell type and context dependent actin-bundling proteins. Our recent work analyzing the role of Fascin during tracheal development in Drosophila has shown that Singed (the Drosophila Fascin homolog) acts as a molecular link between the Branchless (FGF)/Breathless (FGFR) pathway and the actin cytoskeleton. We have reported that the lack of Singed (Sn) leads to wavy and flaccid filopodia due to the disorganization of the tracheal actin cytoskeleton. Here we describe for the first time filopodia breakage in Drosophila, and show that Fascin plays a role in this event. We propose that actin filaments in sn mutant filopodia buckle under membrane pressure due to lower bending stiffness, eventually undergoing breakage. Both Filopodia buckling and breakage would impair correct cell navigation and migration.

MiR-145 overexpression suppresses the migration and invasion of metastatic melanoma cells

MicroRNAs (miRNAs) are post-transcriptional modulators of gene expression which play important roles in tumorigenesis and cancer metastasis. Since they are often highly deregulated in various types of cancer, miRNAs may be effective treatment targets. miRNA profiling studies of melanoma have led to the identification of several tumor suppressor miRNAs. One of these include miR-145, although functional data proving its specific function are limited. Therefore, in this study, we examined the expression levels of miR-145 in three melanoma cell lines (BLM, FM3P and WM793). Additional gain-of-function experiments revealed that miR-145 exerts an anti-proliferative effect in the primary, non-invasive melanoma cell line, WM793, whereas cell migration and the invasive potential of metastatic melanoma cells was suppressed following transfection with miR-145 mimics. In order to investigate the mechanisms by which miR-145 exerts its invasion suppressor function, we examined the expression level of target genes [fascin homolog 1 (FSCN1), myosin‑Va (MYO5A and SOX9] and that of an indirect target (RAB27A) following the overexpression of miR-145. The results showed that SOX9, MYO5A and RAB27A were not involved in the biological effects caused by miR-145 mimics. Surprisingly, we discovered that miR-145 in melanoma, in contrast to many other tumor types, does not necessarily act via the target, FSCN1, since the downregulation of FSCN1 did not inhibit cell proliferation or migration but, on the contrary, increased cell invasion in two out of the three melanoma cell lines examined. Our in vitro data is in accordance with previously reported in vivo data describing the low expression of FSCN1 in malignant melanomas when compared to dysplastic nevi, suggesting that the expression of FSCN1 decreases as the formation and progression stage of melanoma advances. In conclusion, our data provide evidence that miR-145 is an invasion suppressor in metastatic melanoma cells. Despite the fact that it remains unclear which genes or pathways are regulated by miR-145 in melanoma, miR-145 may serve as a useful therapeutic agent in melanoma when re-expressed in situ.

Cutaneous Pigmentation in Health and Disease: Novel and Well-Established Players

Cutaneous pigment formation and aberration in disease are addressed. Dynoodt et al. (this issue) present data on a specific micro RNA that downregulates proteins involved in melanogenesis and melanosome movement. Kim et al. (this issue) present data showing that Wnt signaling is involved in the pathogenesis of melasma. Both articles enhance our understanding of cutaneous pigmentation and point to targets in the development of novel therapeutic modalities.

The Cluster of miR-143 and miR-145 Affects the Risk for Esophageal Squamous Cell Carcinoma through Co-Regulating Fascin Homolog 1

MicroRNAs (miRNAs), 18–24 nt non-coding RNAs, are thought to play important roles in cell proliferation, differentiation, apoptosis, and development. Recent studies suggest that some of the known microRNAs map to a single genomic locale within a single polycistronic transcript. But the roles of the cluster remain to be known. In order to understand the role and mechanism of a cluster of miR-143 and miR-145 in esophageal squamous cell carcinoma (ESCC), the association of mature miR-143 and miR-145 expression with the risk for esophageal cancer was evaluated in ESCC patients with a case-control study, and target protein regulated by mature miRNA was analyzed in ESCC cell lines with 3′UTR luciferase reporter assay. The expression levels of miR-143 and miR-145 were determined in 110 pairs of esophageal cancer tissues and adjacent normal tissues using real-time reverse transcription PCR. The relative expression of miR-143 and miR-145 were statistically different between cancer tissues and matched controls. The combined expression of miR-143 and miR-145 was significantly associated with the risk for esophageal cancer. Meanwhile, the reduced expression of two miRNAs in tumor patient was supposed to have a trend of lymph node metastases. The co-expression pattern of miR-143 and miR-145 was analyzed with Pearson correlation. It showed a significant correlation between these two miRNAs expression both in tissues and tumor cell lines. 3′UTR luciferase reporter assay indicated that Fascin Homolog 1 (FSCN1) could be co-regulated by miR-143 and miR-145. The protein level of FSCN1 showed no significant linear correlation with miR-143 and miR-145 expression in ESCC cell lines with Western blotting analysis. In conclusion, since miR-143 and miR-145 could regulate oncogenic FSCN1 and take part in the modulation of metastases, the result suggested the combination variable of miR-143 and miR-145 as a potential biomarker for earlier diagnosis and prognosis of esophageal cancer.

FSCN1 (fascin homolog 1, actin-bundling protein (Strongylocentrotus purpuratus))

Review on FSCN1 (fascin homolog 1, actin-bundling protein (Strongylocentrotus purpuratus)), with data on DNA, on the protein encoded, and where the gene is implicated.

Abstract 132: miR-133a as a tumor suppressive microRNA targeting multiple oncogenes in head neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinomas (HNSCC) represent 6% of all cancers. Despite of considerable advances in surgery, radiotherapy and chemotherapy, the overall 5-year survival rate for patients is less than 40% in advanced stage. The understanding of the new molecular pathways of HNSCC carcinogenesis would be helpful in improving diagnosis and therapy of the disease. MicroRNA(miRNA) are endogenous short non-coding RNA molecules, which regulate gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner. Growing evidence suggests that miRNAs are aberrantly expressed in many human cancers, and that they play significant roles in carcinogenesis and cancer progression. Bioinformatic prediction indicates that miRNAs regulate more than 30% of the protein coding genes. The study of the tumor suppressive miRNAs and their target genes search is important for understanding of the HNSCC carcinogenesis. Based on our miRNA expression signature of HNSCC, miR-133a is one of the down-regulated miRNA and this finding suggests that miR-133a is a candidate of tumor suppressor. Gain-of-function analysis revealed that miR-133a transfectant inhibited cell proliferation and cell invasion in cancer cells. For target genes search of miR-133a in cancer cells, we performed genome-wide protein coding expression analysis. Among the expression study, several candidate genes were identified, such as, fascin homolog 1 (FSCN1), glutathione S-transferase P1 (GSTP1) and caveolin-1 (CAV1). Silencing of these genes showed that significant inhibition of cell growth and invasion were demonstrated in HNSCC cancer cells. Tumor suppressive miR-133a and its target oncogenes may provide new insights into the mechanisms in cancer. Our findings have therapeutic implications and may be exploited for future HNSCC treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 132. doi:10.1158/1538-7445.AM2011-132

284 MIR-145 FUNCTIONS AS TUMOR SUPPRESSOR AND REGULATES ACTIN-BINDING PROTEINS (FSCN1 AND SWAP70) IN PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: MicroRNAs are small non-coding RNAs that negatively regulate protein-coding genes involved in the development of various malignancies. Down-regulation of miR-145 has been reported in many types of human cancer, including prostate cancer (PCa). In the last meeting, we demonstrated that miR-145 was the most down-regulated microRNA in bladder cancer and had a tumor suppressive function through targeting an actin-binding protein, FSCN1. Increasing evidence indicates that the miR-145 functions as a tumor suppressive miRNA and causes cell viability inhibitions in human cancer cells. However, there is limited knowledge of its targets in PCa. The aim of this study is to identify target genes of miR-145 based on gene expression analysis of miR-145-transfected PCa cells. METHODS: For genome-wide screening for target genes silenced by miR-145 in PCa, we performed oligo-microarray analysis of miR-145 transfectants (PC3, DU145) compared to miR-negative control transfectants. We employed web-based software in order to search for putative target genes of miR-145, and we focused on fascin homolog 1 (FSCN1) and SWAP switching B-cell complex 70kDa subunit (SWAP70), which is also known as F-actin binding protein involved in activating B cell transformation. The luciferase reporter assay was used to confirm the actual binding sites between miR-145 and FSCN1/ SWAP70 mRNA. Cell viability was evaluated by cell proliferation, wound healing, and matrigel invasion assays in si-FSCN1 and siSWAP70 transfectants. A total of 75 clinical prostate specimens were subjected to immunohistochemistry of SWAP70. RESULTS: The mRNA and protein expression of FSCN1 and SWAP70 were markedly repressed in the miR-145 transfectants. The luciferase reporter assay demonstrated that miR-145 was directly bound to the specific sites of FSCN1 and SWAP70 mRNA. Significant inhibitions of cell proliferation, invasion and migration were observed in the si-FSCN1 and the si-SWAP70 transfectants (p 0.0001) as well as the miR-145 transfectants (p 0.0001). The immunohistochemical score of SWAP70 in PCa specimens was significantly higher than that in benign prostate hyperplasia specimens (p 0.0169). CONCLUSIONS: Our data suggest that tumor suppressive miR-145 directory regulates both FSCN1 and SWAP70 that might have oncogenic functions associated with actin-promoting cell motility and malignant transformation in PCa. These pathways may play critical roles in PCa development and serve as a novel therapeutic target in PCa.

Restoration of miR-145 expression suppresses cell proliferation, migration and invasion in prostate cancer by targeting FSCN1

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression, primarily at the post-transcriptional level. Growing evidence suggests that miRNAs function as oncogenes or tumor suppressors in human cancers. The down-regulation of miR-145 has been reported in many types of human cancer, including prostate cancer (PC), suggesting that miR-145 functions as a tumor suppressor. Using the PC cell lines, PC3 and DU145, gain-of-function assays revealed that miR-145 transfection inhibited cell proliferation, migration and invasion. Fascin homolog 1 (FSCN1), an actin-bundling protein, is a candidate target gene of miR-145 based on genome-wide gene expression analysis. A luciferase reporter assay showed a significantly decreased signal at two miR-145 target sites at the 3'UTR of FSCN1, suggesting that miR-145 directly regulates FSCN1. In FSCN1 loss-of-function assays, cell growth, migration and invasion were all inhibited, implying that FSCN1 is associated with the progression of PC. The identification of tumor suppressive miRNAs and their target genes could provide new insights into the potential mechanisms of prostate carcinogenesis.

Functional role of LASP1 in cell viability and its regulation by microRNAs in bladder cancer

ObjectiveOur previous study demonstrated that fascin homolog 1 (FSCN1) might have an oncogenic function in bladder cancer (BC) and that its expression was regulated by specific microRNAs (miRNAs). Recently, LIM and SH3 protein 1 (LASP1) as well as FSCN1 have been reported as actin filament bundling proteins in the same complexes attached to the inner surfaces of cell membranes. We hypothesize that LASP1 as well as FSCN1 have an oncogenic function and that is regulated by miRNAs targeting LASP1 mRNA. MethodsThe expression levels of LASP1 mRNA in 86 clinical samples were evaluated by real-time RT-PCR. LASP1-knockdown BC cell lines were transfected by siRNA in order to examine cellular viability by XTT assay, wound healing assay, and matrigel invasion assay. We employed web-based software in order to search for candidate miRNAs targeting LASP1 mRNA, and we focused on miR-1, miR-133a, miR-145, and miR-218. The luciferase reporter assay was used to confirm the actual binding sites between the miRNAs and LASP1 mRNA. ResultsReal-time RT-PCR showed that LASP1 mRNA expression was higher in 76 clinical BC specimens than in 10 normal bladder epitheliums (P < 0.05). Loss-of-function studies using si-LASP1-transfected BC cell lines demonstrated significant cell viability inhibition (P < 0.0005), cell migration inhibition (P < 0.0001), and a decrease in the number of invading cells (P < 0.005) in the transfectants compared with the controls. Transient transfection of three miRNAs (miR-1, miR-133a, and miR-218), which were predicted as the miRNAs targeting LASP1 mRNA, repressed the expression levels of mRNA and protein levels of LASP1. The luciferase reporter assay demonstrated that the luminescence intensity was significantly decreased in miR-1, miR-133a, and miR-218 transfectants (P < 0.05), suggesting that these miRNAs have actual target sites in the 3′ untranslated region of LASP1 mRNA. Furthermore, significant cell viability inhibitions occurred in miR-218, miR-1, and miR-133a transfectants (P < 0.001). ConclusionOur data indicate that LASP1 may have an oncogenic function and that it might be regulated by miR-1, miR-133a, and miR-218, which may function as tumor suppressive miRNAs in BC.

Abstract 2088: miR-133a as a tumor suppressive microRNA by targeting multiple oncogenes in head and neck squamous cell carcinoma (HNSCC)

Abstract Head and neck squamous cell carcinomas (HNSCC) represent 6% of all cancers. Despite of considerable advances in surgery, radiotherapy and chemotherapy, the overall 5-year survival rate for patients is less than 40% in advanced stage. The understanding of the new molecular pathways of HNSCC carcinogenesis would be helpful in improving diagnosis and therapy of the disease. MicroRNA(miRNA) are endogenous short non-coding RNA molecules, which regulate gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner. miRNA over-expression could result in down-regulation of tumor suppressor genes, whereas their under-expression could lead to oncogenes activation. Expression levels of 665 human mature miRNAs were screened using the stem-loop real-time quantitative PCR method. We identified several down-regulated miRNAs based on miRNA expression signatures in HNSCC. A comparison of miRNA signatures from HSCC and our previous reports (Ichimi et al., Int J Cancer 2009, 125:342-352; Kano et al., AACR 2009, #552; Kikkawa et al., AACR 2009, #587) identified miR-133a that is down-regulated in common, suggesting that miR-133a is candidate tumor suppressor. Gain-of-function analysis revealed that miR-133a transfectant inhibits cell proliferation and cell invasion in cancer cells. For target genes silenced by miR-133a in cancer cells, we performed genome-wide screening (oligo microarray analysis and public database searche). Among the genome-wide analysis, several candidate genes were identified, such as, fascin homolog 1 (FSCN1) and glutathione S-transferase P1 (GSTP1). We focused on FSCN1, an actin-bundling protein, which induces parallel actin bundles in cell protrusions and increases cell motility in several human cancers. The miR-133a which has conserved sequence in the 3′UTR of FSCN1, inhibited of FSCN1 expression. The signal from a luciferase reporter assay was significantly decreased at miR-133a site, suggesting miR-133a directly regulate FSCN1. An FSCN1 loss-of-function assay found significant cell growth and invasion inhibition in HNSCC cells implying an FSCN1 is associated with HSCC carcinogenesis. Tumor suppressive miRNAs and target oncogenes may provide new insights into the mechanisms in cancer. Our findings have therapeutic implications and may be exploited for future HSCC treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2088.

MiR‐145, miR‐133a and miR‐133b: Tumor‐suppressive miRNAs target FSCN1 in esophageal squamous cell carcinoma

MicroRNAs (miRNAs), noncoding RNAs 21-25 nucleotides in length, regulate gene expression primarily at the posttranscriptional level. Growing evidence suggests that miRNAs are aberrantly expressed in many human cancers, and that they play significant roles in carcinogenesis and cancer progression. A search for miRNAs with a tumor-suppressive function in esophageal squamous cell carcinoma (ESCC) was performed using the miRNA expression signatures obtained from ESCC clinical specimens. A subset of 15 miRNAs was significantly downregulated in ESCC. A comparison of miRNA signatures from ESCC and our previous report identified 4 miRNAs that are downregulated in common (miR-145, miR-30a-3p, miR-133a and miR-133b), suggesting that these miRNAs are candidate tumor suppressors. Gain-of-function analysis revealed that 3 transfectants (miR-145, miR-133a and miR-133b) inhibit cell proliferation and cell invasion in ESCC cells. These miRNAs (miR-145, miR-133a and miR-133b), which have conserved sequences in the 3'UTR of FSCN1 (actin-binding protein, Fascin homolog 1), inhibited FSCN1 expression. The signal from a luciferase reporter assay was significantly decreased at 2 miR-145 target sites and 1 miR-133a/b site, suggesting both miRNAs directly regulate FSCN1. An FSCN1 loss-of-function assay found significant cell growth and invasion inhibition, implying an FSCN1 is associated with ESCC carcinogenesis. The identification of tumor-suppressive miRNAs, miR-145, miR-133a and miR-133b, directly control oncogenic FSCN1 gene. These signal pathways of ESCC could provide new insights into potential mechanisms of ESCC carcinogenesis.

Identification of circulating placental mRNA in maternal blood of pregnancies affected with fetal congenital heart diseases at the second trimester of pregnancy: implications for early molecular screening

To investigate whether a significantly aberrant expression of circulating placental mRNA genes related with cardiogenesis can be detected at the second trimester of pregnancy. The study was performed in two stages. First stage (development model group): match of 14 placental tissues at delivery of fetuses with congenital heart disease versus 20 controls. Second stage (validation model group): mRNA amplification of abnormal expressed genes in maternal blood samples from 26 women bearing a fetus with a congenital heart disease matched with 28 controls. We identified four functional categories of genes possibly involved in abnormal heart development: cardiac morphogenesis: tenascin, thioredoxin, salvador homolog 1 protein; extracellular matrix (ECM) and valvular tissue biosynthesis; placental-associated plasma protein, collagen, type I, alpha 2, fibulin-1, heparanase, procollagen-proline, 2-oxoglutarate 4-dioxygenase, alpha polypeptide II, Jumonji, AT rich interactive domain 1B RBP2-like; normal contractile activity: actinin, alpha 4, fascin homolog 1, actin-bundling protein; and congestive heart failure. Altered placental genetic expression was found at term delivery in affected fetuses. The aberration was also confirmed in maternal blood at the second trimester of women bearing a fetus with congenital heart disease. Sensitivity for the most aberrant genes ranged between 42% and 95% at a false positive rate (FPR) of 10%.

Proteomic analysis of stargazer mutant mouse neuronal proteins involved in absence seizure

The stargazer (stg) mutant mouse, having mutation in stargazin, the calcium channel gamma2 subunit, exhibited several neurological disorders including spontaneous absence seizure, cerebellar ataxia, and head tossing. To understand the molecular pathogenic mechanism of the absence seizure resulted from the loss of stargazin function, the thalamic proteomes between control mouse and stg mouse were compared. We identified 12 proteins expressed differentially (> 1.6-fold) by fluorescence two-dimensional difference gel electrophoresis and tandem mass spectrometry. Six of them are involved in basic metabolism including energy metabolism, three in stress response, two in axonal growth regulation, and one in the endoplasmic reticulum processing. All except mortalin showed decreased level of expression in stg mouse. Two stress-related proteins, mouse stress induced phosphoprotein 1 and peroxiredoxin 6 exhibited reduced levels of expression in stg mouse, while the level of another stress protein, mortalin was increased. Analysis of oxidative protein carbonylation in thalamic proteome of stg mouse showed higher level of carbonylated proteins in stg mouse than in control mouse. Interestingly, down-regulation of stress protein mouse stress induced phosphoprotein 1, metabolic enzyme isovaleryl-CoA dehydrogenase, and the two in neuronal axon growth, collapsin response mediator protein 2 and fascin homolog 1 coincides with the results of our previous study on gamma-butyrolactone-induced transient absence seizure. Our results suggest that the pathogenesis mechanism underlying absence seizure may involve the molecular events contributed by these proteins.

Immunohistochemical validation of a novel epithelial and a novel stromal marker of pancreatic ductal adenocarcinoma identified by global expression microarrays: sea urchin fascin homolog and heat shock protein 47.

We extended the results of a previous microarray analysis by immunohistochemical validation of differential protein expression in a series of 57 surgically resected infiltrating ductal pancreatic adenocarcinomas. Two representative genes were examined: sea urchin fascin homolog (overexpressed in both cell lines and primary tumors) and heat shock protein 47 (HSP47; overexpressed in primary tumors only). Protein expression also was evaluated in the precursor lesions of pancreatic cancer pancreatic intraepithelial neoplasia (PanIN), and normal ductal epithelium. Fascin expression was seen in the neoplastic cells of 54 (95%) of 57 ductal adenocarcinomas but not in 49 (94%) of 52 adjacent nonneoplastic epithelium. In the multistep pathogenesis of ductal adenocarcinomas, fascin expression seemed to be a late event, usually present in PanINs 2 and 3. HSP47 expression was almost universal and most intense in the ductal adenocarcinoma-associated stromal desmoplasia (57/57), although 37 cases (65%) also expressed HSP47 in the neoplastic epithelium. HSP47 expression was absent in the majority of nonneoplastic pancreata (46 [88%]). Fascin and HSP47 are novel tumor markers with potential diagnostic and therapeutic implications for pancreatic carcinoma. These results establish the usefulness of global expression platforms to identify novel tumor markers.

Cloning and Expression of a Murine Fascin Homolog from Mouse Brain

The fascins are a widely distributed family of proteins that organize filamentous actin into bundles. We have cloned, sequenced, and expressed the murine homolog. Fascin is most abundant in brain and is found in other tissues including uterus and spleen. The deduced open reading frame encodes a protein of 493 amino acids with a molecular mass of 54,412 Da. Previous solubility problems with bacterially expressed fascins were overcome by producing the mouse protein as a fusion with Escherichia coli thioredoxin. A method for cleaving the fusion protein and for purifying active recombinant fascin is described. The N-terminal sequence and molecular mass estimated on SDS gels indicate that recombinant fascin is full-length. Two-dimensional gel electrophoresis suggests that recombinant fascin is post-translationally modified in a manner similar to that observed in mouse brain. Recombinant fascin and the fusion protein are recognized by monoclonal anti-fascin antibodies and will bundle rabbit skeletal muscle F-actin in vitro at a stoichiometry of 4.1:1 actin to fascin. Electron cryomicroscopy images show that the reconstituted bundles are highly ordered. However, their fine structure differs from that of echinoid fascin-actin bundles. This structural difference can be attributed to fascin.

Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension.

Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin.