A pilot study of the relative number of circulating tumor cells and leukocytes containing actin-binding proteins in head and neck cancer patients

Circulating tumor cells(CTCs)play an important role in tumor metastases,which is positively correlated with an increased risk of death.Actin-binding proteins,including cofilin(CFL1),profilin 1(PFN1),and adenylate cyclase-associated protein 1(CAP1),are thought to be involved in tumor cell motility and metastasis,specifically in head and neck squamous cell carcinoma(HNSCC).However,currently,there are no published studies on CFL1,PFN1,and CAP1 in CTCs and leukocytes in HNSCC patients.We assessed serum levels of CFL1,PFN1,and CAP1 and the number of CTCs and leukocytes containing these proteins in blood from 31 HNSCC patients(T1-4N0-2M0).The analysis used flow cytometry and an enzyme-linked immunosorbent assay kit.We found that CAP1+CTCs and CAP1+leukocyte subpopulations were prevalent in these HNSCC patient samples,while the prevalence rates of CFL1+and PFN1+CTCs were relatively low.Patients with stage T2-4N1-2M0 had CFL1+and PFN1+CTCs with an elevated PFN1 serum level,compared with the T1-3N0M0 group.In summary,the PFN1 serum level and the relative number of PFN1+CD326+CTCs could be valuable prognostic markers for HNSCC metastases.The current study is the first to obtain data regarding the contents of actin-binding proteins(ABPs)in CTCs,and leukocytes in blood from HNSCC patients.This is also the first to assess the relationship between the number of CTCs subgroups and disease characteristics.

Actin-binding Rho activating protein is expressed in the central nervous system of normal adult rats

Previous studies show that actin-binding Rho activating protein （Abra） is expressed in cardiomyocytes and vascular smooth muscle cells. In this study, we investigated the expression profile of Abra in the central nervous system of normal adult rats by confocal immunofluorescence. Results showed that Abra immunostaining was located in neuronal nuclei, cytoplasm and processes in the central nervous system, with the strongest staining in the nuclei; in the cerebral cortex, Abra positive neuronal bodies and processes were distributed in six cortical layers including molecular layer, external granular layer, external pyramidal layer, internal granular layer, internal pyramidal layer and polymorphic layer; in the hippocampus, the cell bodies of Abra positive neurons were distributed evenly in pyramidal layer and granular layer, with positive processes in molecular layer and orien layer; in the cerebellar cortex, Abra staining showed the positive neuronal cell bodies in Purkinje cell layer and granular layer and positive processes in molecular layer; in the spinal cord, Abra-immunopositive products covered the whole gray matter and white matter; co-localization studies showed that Abra was co-stained with F-actin in neuronal cytoplasm and processes, but weakly in the nuclei. In addition, in the hippocampus, Abra was co-stained with F-actin only in neuronal processes, but not in the cell body. This study for the first time presents a comprehensive overview of Abra expression in the central nervous system, providing insights for further investigating the role of Abra in the mature central nervous system.

The distibution of cofilin and DNase I in vivo

Actin is the principal component of the cytoskeleton, a structure that can be disassembled and reassem-bled in a matter of seconds in vivo. The state of assembly of actin in vivo is primarily regulated by one ormore actin binding proteins (ABPs). Typically, the actions of ABPs have been studied one by one, however,we propose that multiple ABPs, acting cooperatively, may be involved in the control of actin filament length.Cofilin and DNase I are two ABPs that have previously been demonstrated to form a ternary complex withactin in vitro. This is the first report to demonstrate their co-localisation in vivo, and differences in theirdistributions. Our observations strongly suggest a physiological role for higher order complexes of actin inregulation of cytoskeletal assembly during processes such as cell division.

Phenotypic analysis of a dwarf and deformed flower3 (ddf3) mutant in rice (Oryza sativa L.) and characterization of candidate genes

Dwarf mutants are the crucial resources for molecular biology research and rice breeding. Here, a rice mutant, dwarf and deformed flower3（ddf3）, was identified in tissue culture of Oryza sativa cv. Dongjin. Compared with wild type, the ddf3 mutant exhibited severe dwarfism, a greater number of tillers and significantly decreased fertility. In addition, leaf length, panicle length, and grain length, were significantly shorter. All internodes of ddf3 were shorter than those of wild type, and histological analysis revealed that internode cell elongation was significantly inhibited in ddf3. In the ddf3 mutant, pollen activity was significantly decreased, and the development of most stigmas was abnormal. Genetic analysis indicated that the ddf3 mutant phenotypes are controlled by a single or tightly linked nuclear genes. Using an F2 mapping population generated from a cross between ddf3 and Yangdao 6（9311）, the DDF3 gene was mapped to a 45.21-kb region between insertion-deletion（In Del） markers M15 and M16 on the long arm of chromosome 7. Sequencing revealed a 13.98-kbdeletion in this region in the ddf3 mutant genome that resulted in the complete or partial deletion of ZF（DHHC type zinc finger protein）, EP（expressed protein）, and FH2（actin-binding FH2 domain-containing protein） genes. Quantitative RT-PCR analyses revealed that in wild type, the transcript levels of FH2 were almost the same in all organs, while ZF was mainly expressed in the panicle, and no expression of EP was detected in any organ. Based on these results, ZF and FH2 could be potential DDF3 candidate genes involved in the regulation of rice morphology and flower organ development. Our work has laid the foundation for future functional analysis of these candidate genes and has provided a profitable gene resource for rice breeding for increased fertility in the future.

An Arabidopsis Class II Formin,AtFH19,Nucleates Actin Assembly,Binds to the Barbed End of Actin Filaments,and Antagonizes the Effect of AtFH1 on Actin Dynamics

Formin is a major protein responsible for regulating the nucleation of actin filaments, and as such, it permits the cell to control where and when to assemble actin arrays. It is encoded by a multigene family comprising 21 members in Arabidopsis thaliana. The Arabidopsis formins can be separated into two phylogenetically-distinct classes： there are 11 class I formins and 10 class II formins. Significant questions remain unanswered regarding the molecular mechanism of actin nucleation and elongation stimulated by each formin isovariant, and how the different isovariants coordinate to regulate actin dynamics in cells. Here, we characterize a class II formin, AtFH19, biochemically. We found that AtFH19 retains all general properties of the formin family, including nucleation and barbed end capping activity. It can also generate actin filaments from a pool of actin monomers bound to profilin. However, both the nucleation and barbed end capping activities of AtFH19 are less efficient compared to those of another well-characterized formin, AtFHI. Interestingly, AtFH19 FH1FH2 competes with AtFH1 FHIFH2 in binding actin filament barbed ends, and inhibits the effect of AtFH1 FHIFH2 on actin. We thus propose a mechanism in which two quantitatively different formins coordinate to regulate actin dynamics by competing for actin filament barbed ends.

Regulation of Actin Dynamics in Pollen Tubes:Control of Actin Polymer Level

Actin cytoskeleton undergoes rapid reorganization in response to internal and external cues. How the dynamics of actin cytoskeleton are regulated, and how its dynamics relate to its function are fundamental questions in plant cell biology. The pollen tube is a well characterized actin-based cell morphogenesis in plants. One of the striking features of actin cytoskeleton characterized in the pollen tube is its surprisingly low level of actin polymer. This special phenomenon might relate to the function of actin cytoskeleton in pollen tubes. Understanding the molecular mechanism underlying this special phenomenon requires careful analysis of actin-binding proteins that modulate actin dynamics directly. Recent biochemical and biophysical analyses of several highly conserved plant actin-binding proteins reveal unusual and unexpected properties, which emphasizes the importance of carefully analyzing their action mechanism and cellular activity. In this review, we highlight an actin monomer sequestering protein, a barbed end capping protein and an F-actin severing and dynamizing protein in plant. We propose that these proteins function in harmony to regulate actin dynamics and maintain the low level of actin polymer in pollen tubes.

ABP41 is Involved in the Pollen Tube Development via Fragmenting Actin Filaments

ABP41 is identified as a novel member of plant villin/gelsolin/fragmin superfamily proteins from lily pollen, which binds stoichiometrically to actin filaments and severs them in vitro. To further understand its in-vivo function and the potential molecular mechanisms, biochemical analysis, fluorescence microscopic observation and microinjection assays were performed. Different biochemical measurements showed that ABP41 maintained actin filaments in forms of short F-actin in vitro. Microinjection of ABP41 into pollen tubes could fragment the pre-existing actin filaments, inhibit the velocity of cytoplasmic streaming, and shorten the length of the clear zone of pollen tube. In addition, it was found that the endogenous ABP41 expressing level was dynamically corresponding to the short actin filament structure in pollen at different stages of pollen germination. Our results suggest that ABP41 is involved in the regulation of actin dynamics during the pollen germination process via maintenance of short dynamic actin filaments.

The Villin/Gelsolin/Fragmin Superfamily Proteins in Plants

The villin/gelsolin/fragmin superfamily is a conserved Ca^2＋-dependent family of actin-regulating proteins that is widely present both in mammalian and non-mammalian organisms. They have traditionally been characterized by the same core of three or six tandem gelsolin subdomains. The study in vertebrates and lower eukaryotic cells has revealed that the villin/gelsolin/fragmin superfamily of proteins has versatile functions including severing, capping, nucleating or bundling actin filaments. In plants, encouraging progress has been made in this field of research in recent years. This review will summarize the identified plant homologs of villin/gelsolin/fragmin superfamily, thus providing a basis for reflection on their biochemical activities and functions in plants.

Actin organization and regulation during pollen tube growth

Pollen is the male gametophyte of seed plants and its tube growth is essential for successful fertilization.Mounting evidence has demonstrated that actin organization and regulation plays a central role in the process of its germination and polarized growth.The native structures and dynamics of actin are subtly modulated by many factors among which numerous actin binding proteins(ABPs)are the most direct and significant regulators.Upstream signals such as Ca^(2+),PIP_(2)(phosphatidylinositol-4,5-bis-phosphate)and GTPases can also indirectly act on actin organization through several ABPs.Under such elaborate regulation,actin structures show dynamically continuous modulation to adapt to the in vivo biologic functions to mediate secretory vesicle transportation and fusion,which lead to normal growth of the pollen tube.Many encouraging progress has been made in the connection between actin regulation and pollen tube growth in recent years.In this review,we summarize different factors that affect actin organization in pollen tube growth and highlight relative research progress.