Syklaasiin assosioituva proteiini (CAP) säätelee aktiini-tukirangan dynamiikkaa solujen liikkumisen ja morfogeneesin yhteydessä.

The highly dynamic remodeling of the actin cytoskeleton is responsible for most motile and morphogenetic processes in all eukaryotic cells. In order to generate appropriate spatial and temporal movements, the actin dynamics must be under tight control of an array of actin binding proteins (ABPs). Many proteins have been shown to play a specific role in actin filament growth or disassembly of older filaments. Very little is known about the proteins affecting recycling i.e. the step where newly depolymerized actin monomers are funneled into new rounds of filament assembly. A central protein family involved in the regulation of actin turnover is cyclase-associated proteins (CAP, called Srv2 in budding yeast). This 50-60 kDa protein was first identified from yeast as a suppressor of an activated RAS-allele and a factor associated with adenylyl cyclase. The CAP proteins harbor N-terminal coiled-coil (cc) domain, originally identified as a site for adenylyl cyclase binding. In the N-terminal half is also a 14-3-3 like domain, which is followed by central proline-rich domains and the WH2 domain. In the C-terminal end locates the highly conserved ADP-G-actin binding domain. In this study, we identified two previously suggested but poorly characterized interaction partners for Srv2/CAP: profilin and ADF/cofilin. Profilins are small proteins (12-16 kDa) that bind ATP-actin monomers and promote the nucleotide exchange of actin. The profilin-ATP-actin complex can be directly targeted to the growth of the filament barbed ends capped by Ena/VASP or formins. ADF/cofilins are also small (13-19 kDa) and highly conserved actin binding proteins. They depolymerize ADP-actin monomers from filament pointed ends and remain bound to ADP-actin strongly inhibiting nucleotide exchange. We revealed that the ADP-actin-cofilin complex is able to directly interact with the 14-3-3 like domain at the N-terminal region of Srv2/CAP. The C-terminal high affinity ADP-actin binding site of Srv2/CAP competes with cofilin for an actin monomer. Cofilin can thus be released from Srv2/CAP for the subsequent round of depolymerization. We also revealed that profilin interacts with the first proline-rich region of Srv2/CAP and that the binding occurs simultaneously with ADP-actin binding to C-terminal domain of Srv2/CAP. Both profilin and Srv2/CAP can promote nucleotide exchange of actin monomer. Because profilin has much higher affinity to ATP-actin than Srv2/CAP, the ATP-actin-profilin complex is released for filament polymerization. While a disruption of cofilin binding in yeast Srv2/CAP produces a severe phenotype comparable to Srv2/CAP deletion, an impairment of profilin binding from Srv2/CAP results in much milder phenotype. This suggests that the interaction with cofilin is essential for the function of Srv2/CAP, whereas profilin can also promote its function without direct interaction with Srv2/CAP. We also show that two CAP isoforms with specific expression patterns are present in mice. CAP1 is the major isoform in most tissues, while CAP2 is predominantly expressed in muscles. Deletion of CAP1 from non-muscle cells results in severe actin phenotype accompanied with mislocalization of cofilin to cytoplasmic aggregates. Together these studies suggest that Srv2/CAP recycles actin monomers from cofilin to profilin and thus it plays a central role in actin dynamics in both yeast and mammalian cells.Kaikista aitotumallisista soluista löytyvä solutukiranka koostuu erilaisista säikeisistä proteiineista, jotka täyttävät solun sisäisen tilan ylläpitäen solun muotoa ja auttaen solua kestämään mekaanista rasitusta. Eräs keskeinen solutukirangan proteiini on nimeltään aktiini. Aktiinilla muodostaa säikeitä, jotka samanaikaisesti kasvavat niin sanotuista plus- ja hajoavat vastakkaisista miinus-päistä. Liikkumattomassa hiivasolussa aktiini-säikeillä on tärkeä tehtävä endosytoosissa, kalvorakkuloiden kuljetuksessa solun sisällä sekä solunjakautumisessa. Eläinsoluissa aktiini osallistuu näiden toimintojen lisäksi solun liikkumiseen. Solun työntyessä eteenpäin kasvavat aktiini-säikeiden plus-päät painavat solukalvoa eteenpäin. Säädelläkseen aktiinitukirangan muutoksia, solulla on laaja kirjo aktiinia sitovia proteiineja. Nämä proteiinit aikaansaavat mm. aktiini-säikeiden rakentumista, hajoamista, haaroittumista ja kiinnittymistä toisiinsa. Näitä ilmiöitä on tutkittu paljon, mutta siitä, miten aktiini-monomeerit saadaan ohjattua kasvaviin aktiini-säikeiden päihin, tiedetään hyvin vähän. Yksi keskeinen aktiinin kierrätykseen osallistuva proteiini on nimeltään syklaasiin assosioituva proteiini, CAP (cyclase-associated protein). Hiivan vastaavasta proteiinista käytetään nimeä Srv2/CAP. CAP proteiinit painavat noin 50-60 kilodaltonia ja koostuvat useista toiminnallisista domeenista. Proteiinin amino-terminaalisessa päässä sijaitsee cc(coiled-coil)-domeeni, jota seuraa 14-3-3-domeeni. Tämän jälkeen proteiinissa on kaksi proliini-rikasta aluetta sekä WH2-domeeni. Karboksi-terminaalisessa domeenissa sijaitsee aiemmin karakterisoitu konservoitunut ADP-aktiinin sitomisalue. Tässä väitöskirjatyössä tutkittiin kahden tunnetun aktiinia sitovan proteiinin, profiliinin ja ADF/kofiliinin, sitoutumista Srv2/CAP:iin, sekä niiden yhteistyötä aktiini-tukirangan dynamiikassa. Profiliinit ovat pieniä (12-15 kDa) ATP-aktiinia sitovia proteiineja, joilla on tärkeä tehtävä aktiiniin sitoutuneen nukleotidin, ADP:n, vaihtamisessa ATP-muotoon. ADF/kofiliinit ovat myös pieniä (13-19 kDa), konservoituneita proteiineja, jotka sitoutuvat voimakkaasti ADP-aktiiniin ja hajottavat aktiinisäikeitä. Tutkimuksessa saimme selville Srv2/CAP:in sitoutuvan suoraan hiivan kofiliiniin, mutta vain, jos tähän on sitoutuneena myös ADP-aktiini. Tuloksemme viittaavat siihen, että tämän jälkeen Srv2/CAP:n karboksi-terminaalinen aktiinia sitoa alue kilpailee ADP-aktiinin kofiliinilta. Koska Srv2/CAP sitoutuu hyvin heikosti pelkkään kofiliiniin, tämä todennäköisesti irtoaa kompleksista ja on taas vapaana toistamaan omaa tehtäväänsä. Totesimme myös profiliinin sitoutuvan Srv2/CAP:in ensimmäiseen proliini-rikkaaseen alueeseen samanaikaisesti kun ADP-aktiini on sitoutuneena Srv2/CAP:iin. Aktiinin nukleotidi vaihdetaan ATP:ksi, joko profiliinin tai Srv2/CAP:n toimesta. Profiliini sitoutuu voimakkaasti ATP-aktiiniin ja irtoaa Srv2/CAP:stä. Vapautuvia Profiliini-ATP-aktiini komplekseja käytetään solussa kontrolloituun aktiini-säikeiden kasvuun. Kofiliinin sitomisen poistaminen Srv2/CAP:sta aiheuttaa hiivassa vakavan fenotyypin, johon liittyy ongelmia mm. solun kasvussa ja aktiinirakenteiden muodostumisessa. Tästä voi päätellä kofiliinin sitomisen olevan hyvin keskeinen ominaisuus Srv2/CAP:in toiminnalle. Tutkimuksissamme hiiren CAP proteiineilla, CAP1:llä ja CAP2:lla, havaitsimme CAP2:n ilmentymisen rajoittuvan lihassoluihin, kun taas CAP1 ilmentyi kaikissa muissa solutyypeissä. CAP1:in poistaminen hiiren melanoomasoluista aiheutti kofiliinin kasaantumisen aktiinin kanssa epänormaaleihin kerääntymiin. Nämä tutkimukset osoittavat CAP:in olevan keskeinen komponentti kofiliinin ja aktiinin kierrätyksessä niin hiiva- kuin eläinsoluissakin

Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density

Intracellular pH is a potent modulator of neuronal functions. By catalyzing (de)hydration of CO2, intracellular carbonic anhydrase (CA(i)) isoforms CA2 and CA7 contribute to neuronal pH buffering and dynamics. The presence of two highly active isoforms in neurons suggests that they may serve isozyme-specific functions unrelated to CO2-(de)hydration. Here, we show that CA7, unlike CA2, binds to filamentous actin, and its overexpression induces formation of thick actin bundles and membrane protrusions in fibroblasts. In CA7-overexpressing neurons, CA7 is enriched in dendritic spines, which leads to aberrant spine morphology. We identified amino acids unique to CA7 that are required for direct actin interactions, promoting actin filament bundling and spine targeting. Disruption of CA7 expression in neocortical neurons leads to higher spine density due to increased proportion of small spines. Thus, our work demonstrates highly distinct subcellular expression patterns of CA7 and CA2, and a novel, structural role of CA7.Peer reviewe

DHCR24 exerts neuroprotection upon inflammation-induced neuronal death

Abstract Background DHCR24, involved in the de novo synthesis of cholesterol and protection of neuronal cells against different stress conditions, has been shown to be selectively downregulated in neurons of the affected brain areas in Alzheimer’s disease. Methods Here, we investigated whether the overexpression of DHCR24 protects neurons against inflammation-induced neuronal death using co-cultures of mouse embryonic primary cortical neurons and BV2 microglial cells upon acute neuroinflammation. Moreover, the effects of DHCR24 overexpression on dendritic spine density and morphology in cultured mature mouse hippocampal neurons and on the outcome measures of ischemia-induced brain damage in vivo in mice were assessed. Results Overexpression of DHCR24 reduced the loss of neurons under inflammation elicited by LPS and IFN-γ treatment in co-cultures of mouse neurons and BV2 microglial cells but did not affect the production of neuroinflammatory mediators, total cellular cholesterol levels, or the activity of proteins linked with neuroprotective signaling. Conversely, the levels of post-synaptic cell adhesion protein neuroligin-1 were significantly increased upon the overexpression of DHCR24 in basal growth conditions. Augmentation of DHCR24 also increased the total number of dendritic spines and the proportion of mushroom spines in mature mouse hippocampal neurons. In vivo, overexpression of DHCR24 in striatum reduced the lesion size measured by MRI in a mouse model of transient focal ischemia. Conclusions These results suggest that the augmentation of DHCR24 levels provides neuroprotection in acute stress conditions, which lead to neuronal loss in vitro and in vivo

Cyclase-associated Protein 1 (CAP1) Promotes Cofilin-induced Actin Dynamics in Mammalian Nonmuscle Cells

Cyclase-associated proteins (CAPs) are highly conserved actin monomer binding proteins present in all eukaryotes. However, the mechanism by which CAPs contribute to actin dynamics has been elusive. In mammals, the situation is further complicated by the presence of two CAP isoforms whose differences have not been characterized. Here, we show that CAP1 is widely expressed in mouse nonmuscle cells, whereas CAP2 is the predominant isoform in developing striated muscles. In cultured NIH3T3 and B16F1 cells, CAP1 is a highly abundant protein that colocalizes with cofilin-1 to dynamic regions of the cortical actin cytoskeleton. Analysis of CAP1 knockdown cells demonstrated that this protein promotes rapid actin filament depolymerization and is important for cell morphology, migration, and endocytosis. Interestingly, depletion of CAP1 leads to an accumulation of cofilin-1 into abnormal cytoplasmic aggregates and to similar cytoskeletal defects to those seen in cofilin-1 knockdown cells, demonstrating that CAP1 is required for proper subcellular localization and function of ADF/cofilin. Together, these data provide the first direct in vivo evidence that CAP promotes rapid actin dynamics in conjunction with ADF/cofilin and is required for several central cellular processes in mammals

Reconstitution and Dissection of the 600-kDa Srv2/CAP Complex: ROLES FOR OLIGOMERIZATION AND COFILIN-ACTIN BINDING IN DRIVING ACTIN TURNOVER*

Srv2/cyclase-associated protein is expressed in virtually all plant, animal, and fungal organisms and has a conserved role in promoting actin depolymerizing factor/cofilin-mediated actin turnover. This is achieved by the abilities of Srv2 to recycle cofilin from ADP-actin monomers and to promote nucleotide exchange (ATP for ADP) on actin monomers. Despite this important and universal role in facilitating actin turnover, the mechanism underlying Srv2 function has remained elusive. Previous studies have demonstrated a critical functional role for the G-actin-binding C-terminal half of Srv2. Here we describe an equally important role in vivo for the N-terminal half of Srv2 in driving actin turnover. We pinpoint this activity to a conserved patch of surface residues on the N-terminal dimeric helical folded domain of Srv2, and we show that this functional site interacts with cofilin-actin complexes. Furthermore, we show that this site is essential for Srv2 acceleration of cofilin-mediated actin turnover in vitro. A cognate Srv2-binding site is identified on a conserved surface of cofilin, suggesting that this function likely extends to other organisms. In addition, our analyses reveal that higher order oligomerization of Srv2 depends on its N-terminal predicted coiled coil domain and that oligomerization optimizes Srv2 function in vitro and in vivo. Based on these data, we present a revised model for the mechanism by which Srv2 promotes actin turnover, in which coordinated activities of its N- and C-terminal halves catalyze sequential steps in recycling cofilin and actin monomers

Additional file: Figure S1. of DHCR24 exerts neuroprotection upon inflammation-induced neuronal death

Overexpression of DHCR24 does not significantly alter the mRNA levels of TNFα, BDNF, HMOX1, and NQO1 in the whole mouse striatum after middle cerebral artery occlusion. GAPDH-normalized TNFα, BDNF, HMOX1, and NQO1 mRNA levels in the whole mouse striatum determined using qPCR do not indicate changes in the striatum between DHCR24 overexpressing and control lentivirus-transduced mice after tMCAO. Mean ± SEM, n = 6. (PDF 228 kb