Regulation of INF2-mediated actin polymerization through site-specific lysine acetylation of actin itself

INF2 is a formin protein that accelerates actin polymerization. A common mechanism for formin regulation is autoinhibition, through interaction between the N-terminal diaphanous inhibitory domain (DID) and C-terminal diaphanous autoregulatory domain (DAD). We recently showed that INF2 uses a variant of this mechanism that we term "facilitated autoinhibition," whereby a complex consisting of cyclase-associated protein (CAP) bound to lysine-acetylated actin (KAc-actin) is required for INF2 inhibition, in a manner requiring INF2-DID. Deacetylation of actin in the CAP/KAc-actin complex activates INF2. Here we use lysine-to-glutamine mutations as acetylmimetics to map the relevant lysines on actin for INF2 regulation, focusing on K50, K61, and K328. Biochemically, K50Q- and K61Q-actin, when bound to CAP2, inhibit full-length INF2 but not INF2 lacking DID. When not bound to CAP, these mutant actins polymerize similarly to WT-actin in the presence or absence of INF2, suggesting that the effect of the mutation is directly on INF2 regulation. In U2OS cells, K50Q- and K61Q-actin inhibit INF2-mediated actin polymerization when expressed at low levels. Direct-binding studies show that the CAP WH2 domain binds INF2-DID with submicromolar affinity but has weak affinity for actin monomers, while INF2-DAD binds CAP/K50Q-actin 5-fold better than CAP/WT-actin. Actin in complex with full-length CAP2 is predominately ATP-bound. These interactions suggest an inhibition model whereby CAP/KAc-actin serves as a bridge between INF2 DID and DAD. In U2OS cells, INF2 is 90-fold and 5-fold less abundant than CAP1 and CAP2, respectively, suggesting that there is sufficient CAP for full INF2 inhibition.Peer reviewe

Large-scale long-term passive-acoustic monitoring reveals spatio-temporal activity patterns of boreal bats

The distribution ranges and spatio-temporal patterns in the occurrence and activity of boreal bats are yet largely unknown due to their cryptic lifestyle and lack of suitable and efficient study methods. We approached the issue by establishing a permanent passive-acoustic sampling setup spanning the area of Finland to gain an understanding on how latitude affects bat species composition and activity patterns in northern Europe. The recorded bat calls were semi-automatically identified for three target taxa; Myotis spp., Eptesicus nilssonii or Pipistrellus nathusii and the seasonal activity patterns were modeled for each taxa across the seven sampling years (2015-2021). We found an increase in activity since 2015 for E. nilssonii and Myotis spp. For E. nilssonii and Myotis spp. we found significant latitude -dependent seasonal activity patterns, where seasonal variation in patterns appeared stronger in the north. Over the years, activity of P. nathusii increased during activity peak in June and late season but decreased in mid season. We found the passive-acoustic monitoring network to be an effective and cost-efficient method for gathering bat activity data to analyze spatio-temporal patterns. Long-term data on the composition and dynamics of bat communities facilitates better estimates of abundances and population trend directions for conservation purposes and predicting the effects of climate change

UNC-45a promotes myosin folding and stress fiber assembly

Contractile actomyosin bundles, stress fibers, are crucial for adhesion, morphogenesis, and mechanosensing in nonmuscle cells. However, the mechanisms by which nonmuscle myosin II (NM-II) is recruited to those structures and assembled into functional bipolar filaments have remained elusive. We report that UNC-45a is a dynamic component of actin stress fibers and functions as a myosin chaperone in vivo. UNC-45a knockout cells display severe defects in stress fiber assembly and consequent abnormalities in cell morphogenesis, polarity, and migration. Experiments combining structured-illumination microscopy, gradient centrifugation, and proteasome inhibition approaches revealed that a large fraction of NM-II and myosin-1c molecules fail to fold in the absence of UNC-45a. The remaining properly folded NM-II molecules display defects in forming functional bipolar filaments. The C-terminal UNC-45/Cro1/She4p domain of UNC-45a is critical for NM-II folding, whereas the N-terminal tetratricopeptide repeat domain contributes to the assembly of functional stress fibers. Thus, UNC-45a promotes generation of contractile actomyosin bundles through synchronized NM-II folding and filament-assembly activities.Peer reviewe

Crystal structure of a tripartite complex between C3dg, C-terminal domains of factor H and OspE of Borrelia burgdorferi

Complement is an important part of innate immunity. The alternative pathway of complement is activated when the main opsonin, C3b coats non-protected surfaces leading to opsonisation, phagocytosis and cell lysis. The alternative pathway is tightly controlled to prevent autoactivation towards host cells. The main regulator of the alternative pathway is factor H (FH), a soluble glycoprotein that terminates complement activation in multiple ways. FH recognizes host cell surfaces via domains 19–20 (FH19-20). All microbes including Borrelia burgdorferi, the causative agent of Lyme borreliosis, must evade complement activation to allow the infectious agent to survive in its host. One major mechanism that Borrelia uses is to recruit FH from host. Several outer surface proteins (Osp) have been described to bind FH via the C-terminus, and OspE is one of them. Here we report the structure of the tripartite complex formed by OspE, FH19-20 and C3dg at 3.18 Å, showing that OspE and C3dg can bind simultaneously to FH19-20. This verifies that FH19-20 interacts via the “common microbial binding site” on domain 20 with OspE and simultaneously and independently via domain 19 with C3dg. The spatial organization of the tripartite complex explains how OspE on the bacterial surface binds FH19-20, leaving FH fully available to protect the bacteria against complement. Additionally, formation of tripartite complex between FH, microbial protein and C3dg might enable enhanced protection, particularly on those regions on the bacteria where previous complement activation led to deposition of C3d. This might be especially important for slow-growing bacteria that cause chronic disease like Borrelia burgdorferi.Peer reviewe

Overall architecture and interfaces of the complex.

<p>(A) C3dg on top (chain A: light orange, chain B: orange, chain C: dark orange), FH19-20 across (chain D: light blue, E: sky blue, F: deep blue) and OspE on the bottom (chain G: pink, I: violet purple). FH is sandwiched between C3dg and OspE and binds C3dg mainly <i>via</i> domain 19 and OspE on the opposing site <i>via</i> domain 20. Position of the thioester and amino acids involved is C3dg is marked using green. (B) A σA weighted electron density map of the binding interface between C3dg and FH19. A cartoon model of C3dg, chain A, is shown in orange on top and FH19-20, chain D, below in blue. Residues involved in binding are shown with electron density around them. (C) A σA weighted electron density map of the binding interface between and FH20 and OspE. FH, chain D is above and OspE, chain G is below. Residues involved in binding are shown as stick model in the electron density.</p

Macromolecular interfaces in the C3dg:FH19-20:OspE complex.

<p>Physiologically relevant interactions are shown with a grey background. A:D:G is trimer 1; B:E:(H) is trimer 2; C:F:I is trimer 3. n.a. = data not available.</p

Binding interface between complement FH19-20 (chain F, grey cartoon) and C3dg (chain C, light yellow surface).

<p>Interface is shown from two directions about 180° apart with the interacting residues shown in ball-and-stick. The interface has 14 hydrogen bonds, marked using dashed lines between FH19 and C3dg. Residues in FH19 are italicised.</p

Binding interface between complement FH19-20 (chain F, grey cartoon) and OspE of <i>Borrelia burgdorferi</i> (chain I, pink surface).

<p>Interface is shown from two directions about 180° apart with the interacting residues shown in ball-and-stick. Binding is mediated <i>via</i> domain 20 of FH19-20. 13 hydrogen bonds in the interface are marked with dashed lines. Residues involved are annotated using a bold font for the amino acids from OspE and normal font for FH.</p

Superimposition of trimers and proteins on each other.

<p>Cα’s of each proteins were compared to each other using align function in PyMol. Values shown are RMSD/Cα expressed in angstroms (Å). In parentheses used vs. total number of Cαs after five cycles is shown. Bold marks comparison of two full trimers.</p

Superimposition of FH19-20 on previously published structures.

<p>FH19 (residues 1104–1164) from FH19-20 structure (PBD 2G71, pink [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188127#pone.0188127.ref018" target="_blank">18</a>] is aligned to chain F (deep blue), the published complex structures with OspE (4J38, yellow) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188127#pone.0188127.ref030" target="_blank">30</a>] and with C3d (2XQW, grey) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188127#pone.0188127.ref023" target="_blank">23</a>]. The orientation of FH20 residues 1201–1515 in the important target recognition loop is clearly different.</p