The biological functions of mouse twinfilin isoforms

The actin cytoskeleton is essential for many cellular processes, including motility, morphogenesis, endocytosis and signal transduction. Actin can exist in monomeric (G-actin) or filamentous (F-actin) form. Actin filaments are considered to be the functional form of actin, generating the protrusive forces characteristic for the actin cytoskeleton. The structure and dynamics of the actin filament and monomer pools are regulated by a large number of actin-binding proteins in eukaryotic cells. Twinfilin is an evolutionarily conserved small actin monomer binding protein. Twinfilin is composed of two ADF/cofilin-like domains, separated by a short linker and followed by a C-terminal tail. Twinfilin forms a stable, high affinity complex with ADP-G-actin, inhibits the nucleotide exchange on actin monomers, and prevents their assembly into filament ends. Twinfilin was originally identified from yeast and has since then been found from all organisms studied except plants. Not much was known about the role of twinfilin in the actin dynamics in mammalian cells before this study. We set out to unravel the mysteries still covering twinfilins functions using biochemistry, cell biology, and genetics. We identified and characterized two mouse isoforms for the previously identified mouse twinfilin-1. The new isoforms, twinfilin-2a and -2b, are generated from the same gene through alternative promoter usage. The three isoforms have distinctive expression patterns, but are similar biochemically. Twinfilin-1 is the major isoform during development and is expressed in high levels in almost all tissues examined. Twinfilin-2a is also expressed almost ubiquitously, but at lower levels. Twinfilin-2b turned out to be a muscle-specific isoform, with very high expression in heart and skeletal muscle. It seems all mouse tissues express at least two twinfilin isoforms, indicating that twinfilins are important regulators of actin dynamics in all cell and tissue types. A knockout mouse line was generated for twinfilin-2a. The mice homozygous for this knockout were viable and developed normally, indicating that twinfilin-2a is dispensable for mouse development. However, it is important to note that twinfilin-2a shows similar expression pattern to twinfilin-1, suggesting that these proteins play redundant roles in mice. All mouse isoforms were shown to be able to sequester actin filaments and have higher affinity for ADP-G-actin than ATP-G-actin. They are also able to directly interact with heterodimeric capping protein and PI(4,5)P2 similar to yeast twinfilin. In this study we also uncovered a novel function for mouse twinfilins; capping actin filament barbed ends. All mouse twinfilin isoforms were shown to possess this function, while yeast and Drosophila twinfilin were not able to cap filament barbed ends. Twinfilins localize to the cytoplasm but also to actin-rich regions in mammalian cells. The subcellular localizations of the isoforms are regulated differently, indicating that even though twinfilins biochemical functions in vitro are very similar, in vivo they can play different roles through different regulatory pathways. Together, this study show that twinfilins regulate actin filament assembly both by sequestering actin monomers and by capping filament barbed ends, and that mammals have three biochemically similar twinfilin isoforms with partially overlapping expression patterns.

Aktiinitukiranka on oleellisen tärkeä useissa solun toiminnoissa, kuten liikkuminen, signaalien välittäminen, endosytoosi ja jakautuminen. Aktiini esiintyy soluissa monomeeri- tai säiemuodossa. Aktiinin toiminnallinen muoto soluissa on säiemäinen. Aktiinisäikeiden rakentumista ja purkamista säätelee suuri joukko solunsisäisiä proteiineja. Twinfiliini on keskeinen proteiini tässä säätelyssä. Twinfiliini paina noin 40 kDa ja koostuu kahdesta ADF-H (homologinen actin depolymerizing factor:lle) domeenista. Twinfiliini muodostaa kompleksin aktiini-monomeerin kanssa, estää nukleotidin vaihtumisen aktiini-monomeerissa ja estää aktiinisäikeiden muodostumisen. Twinfiliini tunnistettiin ensin hiivassa ja on sen jälkeen löydetty kaikista tutkituista eliölajeista, paitsi kasveista. Nisäkkään twinfiliinistä ei tiedetty juuri mitään ennen tämän tutkimuksen aloittamista. Käyttäen solubiologisia, biokemiallisia ja perinnöllisyystieteellisiä menetelmiä olemme pyrkineet selvittämään hiiren twinfiliinin roolia aktiinitukirangan säätelyssä. Tunnistimme ja karakterisoimme kaksi uutta muotoa aiemmin tunnistetulle hiiren twinfiliinille, twinfiliini-1:lle. Uudet muodot, twinfiliini-2a ja -2b, jakavat twinfiliini-1:n biokemialliset ominaisuudet, mutta ilmentyvät eri kudoksissa. Kaikki twinfiliini-muodot sitoutuvat ADP-aktiini-monomeereihin ja aktiinifilamenttien päihin sekä capping proteiiniin. Soluissa twinfiliinit paikallistuvat solulimaan ja aktiini-rikkaisiin alueisiin. Twinfiliinien solunsisäistä sijaintia säädellään eri tavoin, mikä mahdollistaa eri muotojen erilaisen roolin solun toiminnoissa. Twinfiliini-1 on keskeinen muoto yksilönkehityksen aikana ja twinfiliini-2b esiintyy vain lihaskudoksissa. Kaikissa hiiren kudoksissa ilmentyy ainakin kaksi twinfiliinin muotoa, osoittaen, että twinfiliinin täytyy olla tärkeä osatekijä aktiinin säätelyssä. Tutkimuksen aikana loimme poistogeenisen hiirilinjan twinfiliini-2a:n suhteen. Poistogeeniset hiiret olivat elinkelpoisia ja kehittyivät normaalisti. Voi olla, että muut twinfiliini-muodot pystyvät korvaamaan twinfiliini-2a:n toiminnan soluissa.