some mechanisms involved in cell morphogenesis , such as membrane vesicle transport , are conserved at least among crown eukaryotes ( metazoa , fungi and plants ) , whereas others , such as those involving extracellular structures or the precise roles of different rho - like gtpases , are not .
yet other cellular processes , such as cytokinesis , often recruit conserved proteins to accomplish superficially dissimilar tasks ( for example , budding , cleavage or phragmoplast - based cell division of plant cells ) . for many morphogenetic mechanisms ,
the question of evolutionary conservation remains unresolved because available information is limited to one or a few model organisms .
for example , this is the case for the molecular mechanisms that ensure the communication between the cytoskeleton and the surface of the cell .
however , the recent increase in the data available from a number of genome projects allows wide - ranging searches for homologs of known components of signaling and morphogenetic pathways .
the results of such searches can lead both to experimentally testable hypotheses and to general conclusions regarding the evolution of morphogenetic processes .
formins , also known as formin homology ( fh ) proteins , are proteins implicated in cellular and organismal morphogenesis of both metazoa and fungi . on the cellular level , they are involved in the establishment and maintenance of cell and/or tissue polarity , in cytokinesis   and in the positioning of the mitotic spindle .
they interact directly or indirectly with actin , profilin , rho - like gtpases , the yeast spa2 protein and septins , proteins containing sh3 or ww domains , dynein and microtubules .
the yeast formin homolog encoded by bni1 is localized to the cell periphery and participates in positioning cortical actin patches towards distinct regions of the plasma membrane .
some kind of contact with the plasmalemma ( in addition to that mediated by a rho - like gtpase ) might therefore be expected , although there is no evidence as yet for such a contact .
nothing is known about formin function in plants , although the existence of two arabidopsis thaliana proteins containing the conserved formin - homology 2 ( fh2 ) domain has been reported recently .
given that all known formins represent a well - defined family , this class of proteins may be a good candidate for a systematic genome sequence search . here , i present the results of such an approach , which has led to the identification of putative plant formin genes , as well as to the finding that the evolutionarily old formin domain may be used in a number of different ways and contexts ( ' modules ' as defined by hartwell et al . ) by recent eukaryotes .
formins are defined by the presence of two sequence domains - the low - complexity , proline - rich fh1 and the carboxy - terminal fh2 .
a third domain - the amino - terminal fh3 motif - has been characterized biochemically but is rather poorly delimited in sequence terms . despite a conflicting consensus definition , this motif appears to be identical to the amino - terminal conserved block found in some formins by wasserman .
i have used the l - x - x - g - n - x - m - n ( single - letter amino - acid notation ; x is any amino acid ) motif present in the fh2 domain of most fungal and metazoan formins to search for putative arabidopsis formin homologs and found eight such inter - related genes ( see materials and methods and table 1 ) .
all of them correspond either to hypothetical open reading frames ( orfs ) or to unannotated genomic or cdna clones , indicating that at least some of them are expressed in vivo .
these putative genes and their predicted protein products will be referred to henceforth as atformins 1 to 8 .
putative formin - related genes of arabidopsis thaliana   orf coordinates refer to the longest putative orf in the first sequence ( first primary accession ) listed .
sequence comparison with known formins revealed the presence of genuine fh2 domain in all arabidopsis formins ( figure 1 ) .
however , even the longest predicted proteins , encoded by the atformin 3 , -4 and -5 genes , lack parts of the fh2 region ubiquitously conserved among corresponding genes of fungi and metazoa ( figures 1 and 2 ) , although not necessarily among their protein products , because some formin mrnas undergo complex splicing .
sequence motifs corresponding to the missing regions were found in all cases within the predicted introns by visual inspection of three - frame translation data . because the reliability of mrna structure prediction is limited , failure to identify exons correctly may explain the apparent deletion of this region of the fh2 domain .
the possibly mispredicted intron encoding subdomain g of atformin4 is split by a frameshift mutation , however .
although this could reflect a sequencing error , the possibility remains that plant formin homologs have a modular structure within the fh2 domain at the gene level , and that at least some of the fh2-related sequences within predicted introns are vestiges of exons lost by mutation .
red dots denote the position of introns ( not shown in mformin , for which only mrna sequence is available ) .
numbers indicate positions within the sequence and the size of unaligned insertions ; residues corresponding to unambiguous consensus and/or shared by all arabidopsis formins are highlighted . for gene terminology
letters denote subdomain modules with in fh2 as defined in figure 1 . only the ' highly likely ' membrane - spanning segments are shown .
surprisingly , there are two such regions in atformins 2 , 6 and 8-a feature not observed in the non - plant formins examined ( listed in materials and methods ) . neither motifs corresponding to fh3 nor coiled - coil regions flanking fh1 ( common but not ubiquitous in non - plant formins ) were found .
the structure of fh2 , the overall protein size ( smaller than most non - plant formins ) and the domain layout of arabidopsis formins therefore show possible plant - specific features ( figure 2 ) .
this idea is supported by the topology of an evolutionary tree that consistently places arabidopsis formins in a branch separate from other members of the formin family ( figure 3 ) .
unrooted evolutionary tree of fh2 subdomains a , c and h constructed by the neighbor - joining method .
branches in agreement with the tree previously reported by zeller et al .   are highlighted in green , novel branches in yellow .
as in the non - plant formins , the amino - terminal portions of all arabidopsis formins are divergent , although there is 63% identity between atformins 1 and 4 in the overlaping parts of their sequences .
analysis of atformin sequences with smart   revealed no previously characterized domains outside the fh2 region .
however , putative amino - terminal membrane insertion signals ( signal peptides ) followed by a segment highly likely to be membrane - spanning and a variable number of possible transmembrane domains were found in atformins 1 , 2 , 4 , 6 and 8 .
a possible membrane insertion signal was also identified in atformin5 by one of the two methods used ( see materials and methods , and figures 2,4 ) .
the length of predicted signal peptides suggests that they may represent membrane anchors rather than secretion signals .
a putative transmembrane segment was also found in the apparently amino - terminally truncated sequence of atformin3 .
in contrast , no signal peptides were found in 12 fungal and animal formins listed in materials and methods , although transmembrane - like segments were observed in some .
surprisingly , the putative transmembrane segment lies between the two pro - rich regions in atformins 2 , 6 and 8 .
obviously , only the cytoplasmic one of these two motifs can act as a conventional fh1 domain .
its size ranges from 106 to 423 amino acids , with proline content of 13 to 41% and multiple stretches to five to nine consecutive proline residues .
interestingly , the fh1 domains of atformins 2 , 7 and 8 are extremely rich in serine ( up to 20% ) and contain stretches of up to seven consecutive serine residues .   putative membrane anchors and transmembrane domains of arabidopsis formins .
aliphatic ( i , l , v ) , aromatic ( f , h , w , y ) and other potentially hydrophobic ( a , c , g , k , m , r , t ) amino acids are highlighted the other proline - rich domain of atformins 2 , 6 and 8 is predicted to be exposed to a non - cytoplasmic compartment .
given that polyproline stretches are characteristic for a class of structural cell - wall proteins known as extensins , it is tempting to speculate about a possible role for this domain in communication between formins and structures within the cell wall . apart from this ,
although formins are well conserved with respect to their molecular structure , we do not know the extent of their conservation within signaling or structural modules .
as the relationships between protein structure , module structure and biological function are far from straightforward , we can at present neither prove nor exclude the possibility that plant formins contribute to similar functional modules to their animal and fungal counterparts .
the question of whether these proteins have a direct role in cytokinesis , in mitotic spindle localization , or in some other cellular process , possibly involving cytoskeleton rearrangement or cell - surface growth , will have to be answered experimentally .
a systematic search of the available arabidopsis genomic and cdna sequences revealed the presence of eight genes encoding proteins that define a novel subfamily of the formin family .
this indicates a mechanism of membrane localization that may be specific to plants and functionally related to a possible role for formins in the communication between the plant cell and extracellular structures .
the initial search for formin homologues in the non - redundant arabidopsis thaliana protein ( nrat ) database , performed using the patmatch program   with the query pattern l - x - x - g - n - x - m - n , yielded three potential formin homologs - atformin1 to atformin3 .
atformins 2 to 8 were found by a tblastn 2.0 search   in genbank , using the predicted protein sequence of atformin 1 as query ( p(n ) values in the range of 5.810 to 1.310 ) .
known members of the formin family ( a human diaphanous homolog and drosophila melanogaster cappucino ) were found in the same search ( p(n ) values 110 and 1.310 , respectively ) , verifying the statistical significance of the initial patmatch results .  
intron positions in the genomic sequences were determined ( or confirmed ) using the netgene2 server .
all sequence comparisons were done on a set of 20 metazoan , yeast and plant formin sequences . these were fugu , fugu rubripes formin homolog gb|aac34395.1 ; lformin , mouse lymphocyte - specific formin gb|aado1273 ; bnr1 , yeast bnr1 protein sp|p40450 ; bni1 , yeast bni1 protein sp|p4183 ; fhos , human formin - like protein gb|aad39906.1 ; caeno , caenorhabditis elegans formin homolog gb|aab42354.1 ; cappu , d. melanogaster cappuccino gb|aac46925.1 ; p14omdia and p134mdia2 , mouse diaphanous homologs gb|aac53280 and gb|aac71771.1 ; dia - drome , d. melanogaster diaphanous sp|p48608 ; cyk1 , c. elegans cyk1 assembled from gb|aaa81161.1 and gb|aac17501.1 ; mformin , mouse formin sp|qo5860 ; and atformin 1 to 8 .
protein sequences were aligned with the aid of macaw , using the gibbs sampler and segment pair algorithms , blosum45 matrix . only blocks with p<10 were considered .
no homology to fh3 as defined by petersen et al .   or to the amino - terminal conserved region   was revealed by this tool , whereas the fh2 domain was readily identified .
consensus of the resulting alignment of fh2 ( deposited in the embl alignment database , accession number ds39866 ) has been calculated for each subdomain separately ( see figure 1 ) by the method of brown and lai .
the smart program   was used to examine predicted protein sequences for the presence and location of known sequence domains , putative secretion signals , transmembrane segments , coiled - coil motifs and low sequence complexity regions ( usually representing proline - rich fh1 domains whose location was confirmed by visual inspection ) .
prediction of signal peptides by the neural network ( nn ) method ) was independently verified by a hidden markov model - based ( hmm ) method on the signalp 2.0 server ) .
results of both methods were in agreement , with the exception of atformin5 , which was predicted to be membrane - anchored by nn but cytoplasmic by hmm .  
the tree ( figure 3 ) was calculated from the three fh2 subdomains present in all formins studied , using programs from the phylip package   version 3.573 .
an input file was prepared by joining subdomains a , c and h and was used to produce a bootstrapped data set by seqboot with 500 sampling cycles .
distances were calculated using protdist with the pam distance matrix , and the results were used for tree construction using the neighbor - joining method   by neighbor .
the initial search for formin homologues in the non - redundant arabidopsis thaliana protein ( nrat ) database , performed using the patmatch program   with the query pattern l - x - x - g - n - x - m - n , yielded three potential formin homologs - atformin1 to atformin3 .
atformins 2 to 8 were found by a tblastn 2.0 search   in genbank , using the predicted protein sequence of atformin 1 as query ( p(n ) values in the range of 5.810 to 1.310 ) .
known members of the formin family ( a human diaphanous homolog and drosophila melanogaster cappucino ) were found in the same search ( p(n ) values 110 and 1.310 , respectively ) , verifying the statistical significance of the initial patmatch results .  
intron positions in the genomic sequences were determined ( or confirmed ) using the netgene2 server .
all sequence comparisons were done on a set of 20 metazoan , yeast and plant formin sequences . these were fugu , fugu rubripes formin homolog gb|aac34395.1 ; lformin , mouse lymphocyte - specific formin gb|aado1273 ; bnr1 , yeast bnr1 protein sp|p40450 ; bni1 , yeast bni1 protein sp|p4183 ; fhos , human formin - like protein gb|aad39906.1 ; caeno , caenorhabditis elegans formin homolog gb|aab42354.1 ; cappu , d. melanogaster cappuccino gb|aac46925.1 ; p14omdia and p134mdia2 , mouse diaphanous homologs gb|aac53280 and gb|aac71771.1 ; dia - drome , d. melanogaster diaphanous sp|p48608 ; cyk1 , c. elegans cyk1 assembled from gb|aaa81161.1 and gb|aac17501.1 ; mformin , mouse formin sp|qo5860 ; and atformin 1 to 8 .
protein sequences were aligned with the aid of macaw , using the gibbs sampler and segment pair algorithms , blosum45 matrix . only blocks with p<10 were considered .
no homology to fh3 as defined by petersen et al .   or to the amino - terminal conserved region   was revealed by this tool , whereas the fh2 domain was readily identified .
consensus of the resulting alignment of fh2 ( deposited in the embl alignment database , accession number ds39866 ) has been calculated for each subdomain separately ( see figure 1 ) by the method of brown and lai .
the smart program   was used to examine predicted protein sequences for the presence and location of known sequence domains , putative secretion signals , transmembrane segments , coiled - coil motifs and low sequence complexity regions ( usually representing proline - rich fh1 domains whose location was confirmed by visual inspection ) .
prediction of signal peptides by the neural network ( nn ) method ) was independently verified by a hidden markov model - based ( hmm ) method on the signalp 2.0 server ) .
results of both methods were in agreement , with the exception of atformin5 , which was predicted to be membrane - anchored by nn but cytoplasmic by hmm .
the tree ( figure 3 ) was calculated from the three fh2 subdomains present in all formins studied , using programs from the phylip package   version 3.573 .
an input file was prepared by joining subdomains a , c and h and was used to produce a bootstrapped data set by seqboot with 500 sampling cycles .
distances were calculated using protdist with the pam distance matrix , and the results were used for tree construction using the neighbor - joining method   by neighbor .
this work has been supported by the grant agency of the czech republic grant 204/98/0482 and by the czech ministry of education program j13/98:113100003 .
 background : the formin family of proteins has been implicated in signaling pathways of cellular morphogenesis in both animals and fungi ; in the latter case , at least , they participate in communication between the actin cytoskeleton and the cell surface . nevertheless , they appear to be cytoplasmic or nuclear proteins , and it is not clear whether they communicate with the plasma membrane , and if so , how . 
 because nothing is known about formin function in plants , i performed a systematic search for putative arabidopsis thaliana formin homologs.results:i found eight putative formin - coding genes in the publicly available part of the arabidopsis genome sequence and analyzed their predicted protein sequences . 
 surprisingly , some of them lack parts of the conserved formin - homology 2 ( fh2 ) domain and the majority of them seem to have signal sequences and putative transmembrane segments that are not found in yeast or animals formins.conclusions:plant formins define a distinct subfamily . 
 the presence in most arabidopsis formins of sequence motifs typical or transmembrane proteins suggests a mechanism of membrane attachment that may be specific to plant formins , and indicates an unexpected evolutionary flexibility of the conserved formin domain . 
Background
Results and discussion
Conclusions
Materials and methods
Identification of 
Sequence alignment and domain structure analysis
Construction of the evolutionary tree
Acknowledgements