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Henry N Higgs - One of the best experts on this subject based on the ideXlab platform.
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FSGS-Causing INF2 Mutation Impairs Cleaved INF2 N-Fragment Functions in Podocytes.
Journal of the American Society of Nephrology : JASN, 2020Co-Authors: Balajikarthick Subramanian, Henry N Higgs, Johannes Schlondorff, Justin Chun, Paul Yan, Isaac E. Stillman, Seth L. Alper, Chandra Perez-gill, Martin R. PollakAbstract:Background Mutations in the gene encoding inverted formin-2 (INF2), a member of the formin family of actin regulatory proteins, are among the most common causes of autosomal dominant FSGS. INF2 is regulated by interaction between its N-terminal diaphanous inhibitory domain (DID) and its C-terminal diaphanous autoregulatory domain (DAD). INF2 also modulates activity of other formins, such as the mDIA subfamily, and promotes stable microtubule assembly. Why the disease-causing mutations are restricted to the N terminus and how they cause human disease has been unclear. Methods We examined INF2 isoforms present in podocytes and evaluated INF2 cleavage as an explanation for immunoblot findings. We evaluated the expression of INF2 N- and C-terminal fragments in human kidney disease conditions. We also investigated the localization and functions of the DID-containing N-terminal fragment in podocytes and assessed whether the FSGS-associated R218Q mutation impairs INF2 cleavage or the function of the N-fragment. Results The INF2-CAAX isoform is the predominant isoform in podocytes. INF2 is proteolytically cleaved, a process mediated by cathepsin proteases, liberating the N-terminal DID to function independently. Although the N-terminal region normally localizes to podocyte foot processes, it does not do so in the presence of FSGS-associated INF2 mutations. The C-terminal fragment localizes to the cell body irrespective of INF2 mutations. In podocytes, the N-fragment localizes to the plasma membrane, binds mDIA1, and promotes cell spreading in a cleavage-dependent way. The disease-associated R218Q mutation impairs these N-fragment functions but not INF2 cleavage. Conclusions INF2 is cleaved into an N-terminal DID-containing fragment and a C-terminal DAD-containing fragment. Cleavage allows the N-terminal fragment to function independently and helps explain the clustering of FSGS-associated mutations.
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Regulation of INF2-mediated actin polymerization through site-specific lysine acetylation of actin itself.
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Mu A, Tak Shun Fung, Lisa M. Francomacaro, Thao Huynh, Tommi Kotila, Zdenek Svindrych, Henry N HiggsAbstract: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.
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A complex containing lysine-acetylated actin inhibits the formin INF2.
Nature cell biology, 2019Co-Authors: Mu A, Tak Shun Fung, Arminja N. Kettenbach, Rajarshi Chakrabarti, Henry N HiggsAbstract:Inverted formin 2 (INF2) is a member of the formin family of actin assembly factors. Dominant missense mutations in INF2 are linked to two diseases: focal segmental glomerulosclerosis, a kidney disease, and Charcot-Marie-Tooth disease, a neuropathy. All of the disease mutations map to the autoinhibitory diaphanous inhibitory domain. Interestingly, purified INF2 is not autoinhibited, suggesting the existence of other cellular inhibitors. Here, we purified an INF2 inhibitor from mouse brain tissue, and identified it as a complex of lysine-acetylated actin (KAc-actin) and cyclase-associated protein (CAP). Inhibition of INF2 by CAP-KAc-actin is dependent on the INF2 diaphanous inhibitory domain (DID). Treatment of CAP-KAc-actin-inhibited INF2 with histone deacetylase 6 releases INF2 inhibition, whereas inhibitors of histone deacetylase 6 block the activation of cellular INF2. Disease-associated INF2 mutants are poorly inhibited by CAP-KAc-actin, suggesting that focal segmental glomerulosclerosis and Charcot-Marie-Tooth disease result from reduced CAP-KAc-actin binding. These findings reveal a role for KAc-actin in the regulation of an actin assembly factor by a mechanism that we call facilitated autoinhibition.
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Proteolytic program-dependent functions are impaired in INF2-mediated focal segmental glomerulosclerosis.
2019Co-Authors: Balajikarthick Subramanian, Henry N Higgs, Johannes Schlondorff, Justin Chun, Chandra Perez, Paul Yan, Isaac E. Stillman, Seth L. Alper, Martin R. PollakAbstract:Abstract Regulation of the actin cytoskeleton is critical for normal glomerular podocyte structure and function. Altered regulation of the podocyte cytoskeleton can lead to proteinuria, reduced kidney filtration function and focal segmental glomerulosclerosis (FSGS). Mutations in inverted formin 2 (INF2), a member of the formin family of actin regulatory proteins, are the most common cause of autosomal dominant FSGS. INF2 is a multi-domain protein regulated by interaction between its N-terminal Diaphanous Inhibitory Domain (DID) and its C-terminal Diaphanous Auto-regulatory Domain (DAD). Although many aspects of the INF2 DID-DAD interaction are understood, it remains unclear why disease-causing mutations are restricted to the DID and how these mutations cause human disease. Here we report a proteolytic cleavage in INF2 that liberates the INF2 N-terminal DID to function independently of the INF2 C-terminal fragment containing the DAD domain. N-terminal DID region epitopes are differentially localized to podocyte foot process structures in normal glomeruli. This N-terminal fragment localization is lost in INF2-mediated FSGS, whereas INF2 C-terminal fragment epitopes localize to the podocyte cell body in both normal and disease conditions. INF2 cleavage is mediated by cathepsin proteases. In cultured podocytes, the wild-type INF2 N-terminal fragment localizes to membrane regions and promotes cell spreading, while these functions are impaired in a disease-associated INF2 mutant R218Q in the DID. These features are dependent on INF2-cleavage, with accompanying interaction of INF2 N-fragment with mDIA1. Our data suggest a unique cellular function of the DID dependent on INF2 cleavage and help explain the altered localization of FSGS-associated INF2 mutant polypeptides.
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A complex containing lysine-acetylated actin inhibits the formin INF2
2018Co-Authors: Mu A, Tak Shun Fung, Arminja N. Kettenbach, Rajarshi Chakrabarti, Henry N HiggsAbstract:ABSTRACT INF2 is a member of the formin family of actin assembly factors. Dominant mis-sense mutations in INF2 link to two diseases: focal segmental glomerulosclerosis (FSGS), a kidney disease; and Charcot-Marie-Tooth disease (CMTD), a neuropathy. All disease mutations map to the autoinhibitory Diaphanous Inhibitory Domain (DID). Curiously, purified INF2 is not autoinhibited, suggesting the existence of additional cellular inhibitors. We purified an INF2 inhibitor from mouse brain, and identified it as a complex between lysine-acetylated actin (KAc-actin) and cyclase-associated protein (CAP). Inhibition of INF2 by CAP/KAc-actin requires INF2 DID. Treatment of CAP/KAc-actin with histone deacetylase 6 (HDAC6) releases INF2 inhibition, while HDAC6 inhibitors block cellular INF2 activation. INF2 disease mutants are poorly inhibited by CAP/KAc-actin, suggesting that FSGS and CMTD result from reduced CAP/KAc-actin binding. This is the first demonstrated role for lysine-acetylated actin: regulation of an actin assembly factor by a novel mechanism, which we call facilitated auto-inhibition.
Martin R. Pollak - One of the best experts on this subject based on the ideXlab platform.
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FSGS-Causing INF2 Mutation Impairs Cleaved INF2 N-Fragment Functions in Podocytes.
Journal of the American Society of Nephrology : JASN, 2020Co-Authors: Balajikarthick Subramanian, Henry N Higgs, Johannes Schlondorff, Justin Chun, Paul Yan, Isaac E. Stillman, Seth L. Alper, Chandra Perez-gill, Martin R. PollakAbstract:Background Mutations in the gene encoding inverted formin-2 (INF2), a member of the formin family of actin regulatory proteins, are among the most common causes of autosomal dominant FSGS. INF2 is regulated by interaction between its N-terminal diaphanous inhibitory domain (DID) and its C-terminal diaphanous autoregulatory domain (DAD). INF2 also modulates activity of other formins, such as the mDIA subfamily, and promotes stable microtubule assembly. Why the disease-causing mutations are restricted to the N terminus and how they cause human disease has been unclear. Methods We examined INF2 isoforms present in podocytes and evaluated INF2 cleavage as an explanation for immunoblot findings. We evaluated the expression of INF2 N- and C-terminal fragments in human kidney disease conditions. We also investigated the localization and functions of the DID-containing N-terminal fragment in podocytes and assessed whether the FSGS-associated R218Q mutation impairs INF2 cleavage or the function of the N-fragment. Results The INF2-CAAX isoform is the predominant isoform in podocytes. INF2 is proteolytically cleaved, a process mediated by cathepsin proteases, liberating the N-terminal DID to function independently. Although the N-terminal region normally localizes to podocyte foot processes, it does not do so in the presence of FSGS-associated INF2 mutations. The C-terminal fragment localizes to the cell body irrespective of INF2 mutations. In podocytes, the N-fragment localizes to the plasma membrane, binds mDIA1, and promotes cell spreading in a cleavage-dependent way. The disease-associated R218Q mutation impairs these N-fragment functions but not INF2 cleavage. Conclusions INF2 is cleaved into an N-terminal DID-containing fragment and a C-terminal DAD-containing fragment. Cleavage allows the N-terminal fragment to function independently and helps explain the clustering of FSGS-associated mutations.
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Proteolytic program-dependent functions are impaired in INF2-mediated focal segmental glomerulosclerosis.
2019Co-Authors: Balajikarthick Subramanian, Henry N Higgs, Johannes Schlondorff, Justin Chun, Chandra Perez, Paul Yan, Isaac E. Stillman, Seth L. Alper, Martin R. PollakAbstract:Abstract Regulation of the actin cytoskeleton is critical for normal glomerular podocyte structure and function. Altered regulation of the podocyte cytoskeleton can lead to proteinuria, reduced kidney filtration function and focal segmental glomerulosclerosis (FSGS). Mutations in inverted formin 2 (INF2), a member of the formin family of actin regulatory proteins, are the most common cause of autosomal dominant FSGS. INF2 is a multi-domain protein regulated by interaction between its N-terminal Diaphanous Inhibitory Domain (DID) and its C-terminal Diaphanous Auto-regulatory Domain (DAD). Although many aspects of the INF2 DID-DAD interaction are understood, it remains unclear why disease-causing mutations are restricted to the DID and how these mutations cause human disease. Here we report a proteolytic cleavage in INF2 that liberates the INF2 N-terminal DID to function independently of the INF2 C-terminal fragment containing the DAD domain. N-terminal DID region epitopes are differentially localized to podocyte foot process structures in normal glomeruli. This N-terminal fragment localization is lost in INF2-mediated FSGS, whereas INF2 C-terminal fragment epitopes localize to the podocyte cell body in both normal and disease conditions. INF2 cleavage is mediated by cathepsin proteases. In cultured podocytes, the wild-type INF2 N-terminal fragment localizes to membrane regions and promotes cell spreading, while these functions are impaired in a disease-associated INF2 mutant R218Q in the DID. These features are dependent on INF2-cleavage, with accompanying interaction of INF2 N-fragment with mDIA1. Our data suggest a unique cellular function of the DID dependent on INF2 cleavage and help explain the altered localization of FSGS-associated INF2 mutant polypeptides.
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human kidney disease causing INF2 mutations perturb rho dia signaling in the glomerulus
EBioMedicine, 2014Co-Authors: Martin R. Pollak, Khaldoun I Alromaih, Calum A MacraeAbstract:Mutations in Inverted Formin 2 (INF2), a diaphanous formin family protein that regulates actin cytoskeleton dynamics, cause focal segmental glomerulosclerosis (FSGS) and Charcot–Marie–Tooth Disease (CMT) in humans. In addition to directly remodeling actin filaments in vitro, we have shown that INF2 regulates intracellular actin dynamics and actin dependent cellular behavior by opposing Rhoa/Dia signaling. As a step towards a better understanding of the human kidney disease, we wanted to explore the relevance of these findings to the in vivo situation. We used dose dependent knockdown of INF2 to first define an in vivo model and establish an overt glomerular phenotype in zebrafish. This simple assay was validated by rescue with wild type INF2 confirming the specificity of the findings. The edema, podocyte dysfunction, and an altered glomerular filtration barrier observed in the zebrafish pronephros correlate with mistrafficking of glomerular slit diaphragm proteins, defective slit-diaphragm signaling, and disinhibited diaphanous formin (mDia) activity. In contrast to wild-type human INF2, INF2 mutants associated with kidney disease fail to rescue the zINF2 morphant phenotype. Of particular interest, this INF2 knockdown phenotype is also rescued by loss of either RhoA or Dia2. This simple assay allows the demonstration that INF2 functions, at least in part, to modulate Dia-mediated Rho signaling, and that disease causing mutations specifically impair this regulatory function. These data support a model in which disease-associated diaphanous inhibitory domain (DID) mutants in INF2 interfere with its binding to and inhibition of Dia, leading to uncontrolled Rho/Dia signaling and perturbed actin dynamics. Methods to fine tune Rho signaling in the glomerulus may lead to new approaches to therapy in humans.
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Human Kidney Disease-causing INF2 Mutations Perturb Rho/Dia Signaling in the Glomerulus.
EBioMedicine, 2014Co-Authors: Hua Sun, Calum A Macrae, Khaldoun I Al-romaih, Martin R. PollakAbstract:Mutations in Inverted Formin 2 (INF2), a diaphanous formin family protein that regulates actin cytoskeleton dynamics, cause focal segmental glomerulosclerosis (FSGS) and Charcot-Marie-Tooth Disease (CMT) in humans. In addition to directly remodeling actin filaments in vitro, we have shown that INF2 regulates intracellular actin dynamics and actin dependent cellular behavior by opposing Rhoa/Dia signaling. As a step towards a better understanding of the human kidney disease, we wanted to explore the relevance of these findings to the in vivo situation. We used dose dependent knockdown of INF2 to first define an in vivo model and establish an overt glomerular phenotype in zebrafish. This simple assay was validated by rescue with wild type INF2 confirming the specificity of the findings. The edema, podocyte dysfunction, and an altered glomerular filtration barrier observed in the zebrafish pronephros correlate with mistrafficking of glomerular slit diaphragm proteins, defective slit-diaphragm signaling, and disinhibited diaphanous formin (mDia) activity. In contrast to wild-type human INF2, INF2 mutants associated with kidney disease fail to rescue the zINF2 morphant phenotype. Of particular interest, this INF2 knockdown phenotype is also rescued by loss of either RhoA or Dia2. This simple assay allows the demonstration that INF2 functions, at least in part, to modulate Dia-mediated Rho signaling, and that disease causing mutations specifically impair this regulatory function. These data support a model in which disease-associated diaphanous inhibitory domain (DID) mutants in INF2 interfere with its binding to and inhibition of Dia, leading to uncontrolled Rho/Dia signaling and perturbed actin dynamics. Methods to fine tune Rho signaling in the glomerulus may lead to new approaches to therapy in humans.
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Inverted Formin 2 Regulates Actin Dynamics by Antagonizing Rho/Diaphanous-related Formin Signaling
Journal of the American Society of Nephrology : JASN, 2013Co-Authors: Hua Sun, Henry N Higgs, Johannes Schlondorff, Martin R. PollakAbstract:Mutations in inverted formin 2 INF2 are a common cause of familial FSGS. INF2 interacts with diaphanous-related formins (mDia) and antagonizes mDia-mediated actin polymerization in response to active Rho signaling, suggesting that dysregulation of these pathways may mediate the development of INF2-related FSGS. However, the precise mechanisms by which INF2 regulates actin-dependent podocyte behavior remain largely unknown. Here, we investigated the possible role of INF2 in both lamellipodia-associated actin dynamics and actin-dependent slit diaphragm (SD) protein trafficking by manipulating the expression of INF2 and the activity of Rho/mDia signaling in cultured podocytes. Activation of mDia in the absence of INF2 led to defective formation of lamellipodia and abnormal SD trafficking. Effects of mutations disrupting the INF2-mDia interaction suggested the specificity of the mDia-antagonizing effect of INF2 in maintaining the lamellipodium. Furthermore, we found that SD trafficking requires INF2 interaction with lipid raft components. In summary, INF2 regulates lamellipodial actin dynamics and the trafficking of slit diaphragm proteins by opposing Rho/mDia-mediated actin polymerization. Thus, in podocytes, INF2 appears to be an important modulator of actin-dependent behaviors that are under the control of Rho/mDia signaling.
Hua Sun - One of the best experts on this subject based on the ideXlab platform.
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Mice with mutant INF2 show impaired podocyte and slit diaphragm integrity in response to protamine-induced kidney injury
Kidney international, 2016Co-Authors: Balajikarthick Subramanian, Henry N Higgs, Hua Sun, Paul Yan, Victoria T. Charoonratana, Fang Wang, Ka Man V Lai, David M. Valenzuela, Elizabeth J. Brown, Johannes SchlondorffAbstract:Mutations in the INF2 (inverted formin 2) gene, encoding a diaphanous formin family protein that regulates actin cytoskeleton dynamics, cause human focal segmental glomerulosclerosis (FSGS). INF2 interacts directly with certain other mammalian diaphanous formin proteins (mDia) that function as RhoA effector molecules. FSGS-causing INF2 mutations impair these interactions and disrupt the ability of INF2 to regulate Rho/Dia-mediated actin dynamics in vitro . However, the precise mechanisms by which INF2 regulates and INF2 mutations impair glomerular structure and function remain unknown. Here, we characterize an INF2 R218Q point-mutant (knockin) mouse to help answer these questions. Knockin mice have no significant renal pathology or proteinuria at baseline despite diminished INF2 protein levels. INF2 mutant podocytes do show impaired reversal of protamine sulfate–induced foot process effacement by heparin sulfate perfusion. This is associated with persistent podocyte cytoplasmic aggregation, nephrin phosphorylation, and nephrin and podocin mislocalization, as well as impaired recovery of mDia membrane localization. These changes were partially mimicked in podocyte outgrowth cultures, in which podocytes from knockin mice show altered cellular protrusions compared to those from wild-type mice. Thus, in mice, normal INF2 function is not required for glomerular development but normal INF2 is required for regulation of the actin-based behaviors necessary for response to and/or recovery from injury.
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Human Kidney Disease-causing INF2 Mutations Perturb Rho/Dia Signaling in the Glomerulus.
EBioMedicine, 2014Co-Authors: Hua Sun, Calum A Macrae, Khaldoun I Al-romaih, Martin R. PollakAbstract:Mutations in Inverted Formin 2 (INF2), a diaphanous formin family protein that regulates actin cytoskeleton dynamics, cause focal segmental glomerulosclerosis (FSGS) and Charcot-Marie-Tooth Disease (CMT) in humans. In addition to directly remodeling actin filaments in vitro, we have shown that INF2 regulates intracellular actin dynamics and actin dependent cellular behavior by opposing Rhoa/Dia signaling. As a step towards a better understanding of the human kidney disease, we wanted to explore the relevance of these findings to the in vivo situation. We used dose dependent knockdown of INF2 to first define an in vivo model and establish an overt glomerular phenotype in zebrafish. This simple assay was validated by rescue with wild type INF2 confirming the specificity of the findings. The edema, podocyte dysfunction, and an altered glomerular filtration barrier observed in the zebrafish pronephros correlate with mistrafficking of glomerular slit diaphragm proteins, defective slit-diaphragm signaling, and disinhibited diaphanous formin (mDia) activity. In contrast to wild-type human INF2, INF2 mutants associated with kidney disease fail to rescue the zINF2 morphant phenotype. Of particular interest, this INF2 knockdown phenotype is also rescued by loss of either RhoA or Dia2. This simple assay allows the demonstration that INF2 functions, at least in part, to modulate Dia-mediated Rho signaling, and that disease causing mutations specifically impair this regulatory function. These data support a model in which disease-associated diaphanous inhibitory domain (DID) mutants in INF2 interfere with its binding to and inhibition of Dia, leading to uncontrolled Rho/Dia signaling and perturbed actin dynamics. Methods to fine tune Rho signaling in the glomerulus may lead to new approaches to therapy in humans.
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Inverted Formin 2 Regulates Actin Dynamics by Antagonizing Rho/Diaphanous-related Formin Signaling
Journal of the American Society of Nephrology : JASN, 2013Co-Authors: Hua Sun, Henry N Higgs, Johannes Schlondorff, Martin R. PollakAbstract:Mutations in inverted formin 2 INF2 are a common cause of familial FSGS. INF2 interacts with diaphanous-related formins (mDia) and antagonizes mDia-mediated actin polymerization in response to active Rho signaling, suggesting that dysregulation of these pathways may mediate the development of INF2-related FSGS. However, the precise mechanisms by which INF2 regulates actin-dependent podocyte behavior remain largely unknown. Here, we investigated the possible role of INF2 in both lamellipodia-associated actin dynamics and actin-dependent slit diaphragm (SD) protein trafficking by manipulating the expression of INF2 and the activity of Rho/mDia signaling in cultured podocytes. Activation of mDia in the absence of INF2 led to defective formation of lamellipodia and abnormal SD trafficking. Effects of mutations disrupting the INF2-mDia interaction suggested the specificity of the mDia-antagonizing effect of INF2 in maintaining the lamellipodium. Furthermore, we found that SD trafficking requires INF2 interaction with lipid raft components. In summary, INF2 regulates lamellipodial actin dynamics and the trafficking of slit diaphragm proteins by opposing Rho/mDia-mediated actin polymerization. Thus, in podocytes, INF2 appears to be an important modulator of actin-dependent behaviors that are under the control of Rho/mDia signaling.
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Mutations in the INF2 gene account for a significant proportion of familial but not sporadic focal and segmental glomerulosclerosis
Kidney international, 2012Co-Authors: Moumita Barua, Hua Sun, Victoria T. Charoonratana, Elizabeth J. Brown, Giulio Genovese, Martin R. PollakAbstract:Mutations in the inverted formin 2 gene ( INF2 ) have recently been identified as the most common cause of autosomal dominant focal and segmental glomerulosclerosis (FSGS). To quantify the contribution of various genes contributing to FSGS, we sequenced INF2 where all mutations have previously been described (exons 2 to 5) in a total of 215 probands and 281 sporadic individuals with FSGS, along with other known genes accounting for autosomal dominant FSGS ( ACTN4 , TRPC6 , and CD2AP ) in 213 probands. Variants were classified as disease-causing if they altered the amino acid sequence and if they were not found in control samples and in families segregated with disease. Mutations in INF2 were found in a total of 20 of the 215 families (including those previously reported) in our cohort of autosomal dominant familial nephrotic syndrome or FSGS, thereby explaining disease in 9%. INF2 mutations were found in 2 of 281 individuals with sporadic FSGS. In contrast, ACTN4 - and TRPC6 -related diseases accounted for 3 and 2% of our familial cohort, respectively. INF2 -related disease showed variable penetrance, with onset of disease ranging widely from childhood to adulthood, and commonly leading to end-stage renal disease in the third and fourth decade of life. Thus, mutations in INF2 are a more common, although still a minor, monogenic cause of familial FSGS when compared with other known autosomal dominant genes associated with FSGS.
Pinar S. Gurel - One of the best experts on this subject based on the ideXlab platform.
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assembly and turnover of short actin filaments by the formin INF2 and profilin
Journal of Biological Chemistry, 2015Co-Authors: Pinar S. Gurel, Bingquian Guo, Rui Shu, Dale F. Mierke, Henry N HiggsAbstract:INF2 (inverted formin 2) is a formin protein with unique biochemical effects on actin. In addition to the common formin ability to accelerate actin nucleation and elongation, INF2 can also sever filaments and accelerate their depolymerization. Although we understand key attributes of INF2-mediated severing, we do not understand the mechanism by which INF2 accelerates depolymerization subsequent to severing. Here, we show that INF2 can create short filaments (<60 nm) that continuously turn over actin subunits through a combination of barbed end elongation, severing, and WH2 motif-mediated depolymerization. This pseudo-steady state condition occurs whether starting from actin filaments or monomers. The rate-limiting step of the cycle is nucleotide exchange of ADP for ATP on actin monomers after release from the INF2/actin complex. Profilin addition has two effects: 1) to accelerate filament turnover 6-fold by accelerating nucleotide exchange and 2) to shift the equilibrium toward polymerization, resulting in longer filaments. In sum, our findings show that the combination of multiple interactions of INF2 with actin can work in concert to increase the ATP turnover rate of actin. Depending on the ratio of INF2:actin, this increased flux can result in rapid filament depolymerization or maintenance of short filaments. We also show that high concentrations of cytochalasin D accelerate ATP turnover by actin but through a different mechanism from that of INF2.
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Assembly and turnover of short actin filaments by the formin INF2 and profilin.
The Journal of biological chemistry, 2015Co-Authors: Pinar S. Gurel, Mu A, Bingquian Guo, Rui Shu, Dale F. Mierke, Henry N HiggsAbstract:Abstract Inverted Formin 2 (INF2) is a formin protein with unique biochemical effects on actin. In addition to the common formin ability to accelerate actin nucleation and elongation, INF2 can also sever filaments and accelerate their depolymerization. While we understand key attributes of INF2-mediated severing, we do not understand the mechanism by which INF2 accelerates depolymerization subsequent to severing. Here, we show that INF2 can create short filaments (< 60 nm) that continuously turnover actin subunits through a combination of barbed end elongation, severing, and WH2 motif-mediated depolymerization. This pseudo-steady state condition occurs whether starting from actin filaments or monomers. The rate-limiting step of the cycle is nucleotide exchange of ADP for ATP on actin monomers after release from the INF2/actin complex. Profilin addition has two effects: 1) to accelerate filament turnover six-fold by accelerating nucleotide exchange; and 2) to shift the equilibrium toward polymerization, resulting in longer filaments. In sum, our findings show that the combination of multiple interactions between INF2 and actin can work in concert to increase ATP turnover rate. Depending on the ratio of INF2:actin, this increased flux can result in rapid filament depolymerization or maintenance of short filaments. We also show that high concentrations of cytochalasin D accelerate ATP turnover by actin, but through a different mechanism from that of INF2.
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nanostructured self assembly of inverted formin 2 INF2 and f actin INF2 complexes revealed by atomic force microscopy
Langmuir, 2014Co-Authors: Shivani Sharma, Henry N Higgs, Pinar S. Gurel, Elena E Grintsevich, Emil Reisler, Jungreem Woo, James K. GimzewskiAbstract:Self-organization of cytoskeletal proteins such as actin and tubulin into filaments and microtubules is frequently assisted by the proteins binding to them. Formins are regulatory proteins that nucleate the formation of new filaments and are essential for a wide range of cellular functions. The vertebrate inverted formin 2 (INF2) has both actin filament nucleating and severing/depolymerizing activities connected to its ability to encircle actin filaments. Using atomic force microscopy, we report that a formin homology 2 (FH2) domain-containing construct of INF2 (INF2-FH1-FH2-C or INF2-FFC) self-assembles into nanoscale ringlike oligomeric structures in the absence of actin filaments, demonstrating an inherent ability to reorganize from a dimeric to an oligomeric state. A construct lacking the C-terminal region (INF2-FH1-FH2 or INF2-FF) also oligomerizes, confirming the dominant role of FH2-mediated interactions. Moreover, INF2-FFC domains were observed to organize into ringlike structures around single actin filaments. This is the first demonstration that formin FH2 domains can self-assemble into oligomers in the absence of filaments and has important implications for observing unaveraged decoration and/or remodeling of filaments by actin binding proteins.
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Nanostructured Self-Assembly of Inverted Formin 2 (INF2) and F-Actin–INF2 Complexes Revealed by Atomic Force Microscopy
Langmuir : the ACS journal of surfaces and colloids, 2014Co-Authors: Shivani Sharma, Henry N Higgs, Pinar S. Gurel, Elena E Grintsevich, Emil Reisler, Jungreem Woo, James K. GimzewskiAbstract:Self-organization of cytoskeletal proteins such as actin and tubulin into filaments and microtubules is frequently assisted by the proteins binding to them. Formins are regulatory proteins that nucleate the formation of new filaments and are essential for a wide range of cellular functions. The vertebrate inverted formin 2 (INF2) has both actin filament nucleating and severing/depolymerizing activities connected to its ability to encircle actin filaments. Using atomic force microscopy, we report that a formin homology 2 (FH2) domain-containing construct of INF2 (INF2-FH1-FH2-C or INF2-FFC) self-assembles into nanoscale ringlike oligomeric structures in the absence of actin filaments, demonstrating an inherent ability to reorganize from a dimeric to an oligomeric state. A construct lacking the C-terminal region (INF2-FH1-FH2 or INF2-FF) also oligomerizes, confirming the dominant role of FH2-mediated interactions. Moreover, INF2-FFC domains were observed to organize into ringlike structures around single actin filaments. This is the first demonstration that formin FH2 domains can self-assemble into oligomers in the absence of filaments and has important implications for observing unaveraged decoration and/or remodeling of filaments by actin binding proteins.
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INF2-Mediated Severing through Actin Filament Encirclement and Disruption.
Current Biology - CB, 2014Co-Authors: Pinar S. Gurel, Laurent Blanchoin, Rui Shu, Elena E Grintsevich, Z Hong Zhou, Emil Reisler, Henry N HiggsAbstract:BACKGROUND: INF2 is a formin protein with the unique ability to accelerate both actin polymerization and depolymerization, the latter requiring filament severing. Mutations in INF2 lead to the kidney disease focal segmental glomerulosclerosis (FSGS) and the neurological disorder Charcot-Marie Tooth disease (CMTD). RESULTS: Here, we compare the severing mechanism of INF2 with that of the well-studied severing protein cofilin. INF2, like cofilin, binds stoichiometrically to filament sides and severs in a manner that requires phosphate release from the filament. In contrast to cofilin, however, INF2 binds ADP and ADP-Pi filaments equally well. Furthermore, two-color total internal reflection fluorescence (TIRF) microscopy reveals that a low number of INF2 molecules, as few as a single INF2 dimer, are capable of severing, while measurable cofilin-mediated severing requires more extensive binding. Hence, INF2 is a more potent severing protein than cofilin. While a construct containing the FH1 and FH2 domains alone has some severing activity, addition of the C-terminal region increases severing potency by 40-fold, and we show that the WH2-resembling DAD motif is responsible for this increase. Helical 3D reconstruction from electron micrographs at 20 Å resolution provides a structure of filament-bound INF2, showing that the FH2 domain encircles the filament. CONCLUSIONS: We propose a severing model in which FH2 binding and phosphate release causes local filament deformation, allowing the DAD to bind adjacent actin protomers, further disrupting filament structure.
Miguel A Alonso - One of the best experts on this subject based on the ideXlab platform.
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The formin INF2 in disease: progress from 10 years of research.
Cellular and molecular life sciences : CMLS, 2020Co-Authors: Leticia Labat-de-hoz, Miguel A AlonsoAbstract:Formins are a conserved family of proteins that primarily act to form linear polymers of actin. Despite their importance to the normal functioning of the cytoskeleton, for a long time, the only two formin genes known to be a genetic cause of human disorders were DIAPH1 and DIAPH3, whose mutation causes two distinct forms of hereditary deafness. In the last 10 years, however, the formin INF2 has emerged as an important target of mutations responsible for the appearance of focal segmental glomerulosclerosis, which are histological lesions associated with glomerulus degeneration that often leads to end-stage renal disease. In some rare cases, focal segmental glomerulosclerosis concurs with Charcot-Marie-Tooth disease, which is a degenerative neurological disorder affecting peripheral nerves. All known INF2 gene mutations causing disease map to the exons encoding the amino-terminal domain. In this review, we summarize the structure, biochemical features and functions of INF2, conduct a systematic and comprehensive analysis of the pathogenic INF2 mutations, including a detailed study exon-by-exon of patient cases and mutations, address the impact of the pathogenic mutations on the structure, regulation and known functions of INF2, draw a series of conclusions that could be useful for INF2-related disease diagnosis, and suggest lines of research for future work on the molecular mechanisms by which INF2 causes disease.
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The formin INF2 in disease: progress from 10 years of research
Cellular and Molecular Life Sciences, 2020Co-Authors: Leticia Labat-de-hoz, Miguel A AlonsoAbstract:Formins are a conserved family of proteins that primarily act to form linear polymers of actin. Despite their importance to the normal functioning of the cytoskeleton, for a long time, the only two formin genes known to be a genetic cause of human disorders were DIAPH1 and DIAPH3 , whose mutation causes two distinct forms of hereditary deafness. In the last 10 years, however, the formin INF2 has emerged as an important target of mutations responsible for the appearance of focal segmental glomerulosclerosis, which are histological lesions associated with glomerulus degeneration that often leads to end-stage renal disease. In some rare cases, focal segmental glomerulosclerosis concurs with Charcot–Marie–Tooth disease, which is a degenerative neurological disorder affecting peripheral nerves. All known INF2 gene mutations causing disease map to the exons encoding the amino-terminal domain. In this review, we summarize the structure, biochemical features and functions of INF2, conduct a systematic and comprehensive analysis of the pathogenic INF2 mutations, including a detailed study exon-by-exon of patient cases and mutations, address the impact of the pathogenic mutations on the structure, regulation and known functions of INF2, draw a series of conclusions that could be useful for INF2-related disease diagnosis, and suggest lines of research for future work on the molecular mechanisms by which INF2 causes disease.
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The actin-MRTF-SRF transcriptional circuit controls tubulin acetylation via α-TAT1 gene expression.
The Journal of cell biology, 2018Co-Authors: Jaime Fernández-barrera, Miguel Bernabé-rubio, Javier Casares-arias, Laura Rangel, Laura Fernández-martín, Isabel Correas, Miguel A AlonsoAbstract:The role of formins in microtubules is not well understood. In this study, we have investigated the mechanism by which INF2, a formin mutated in degenerative renal and neurological hereditary disorders, controls microtubule acetylation. We found that silencing of INF2 in epithelial RPE-1 cells produced a dramatic drop in tubulin acetylation, increased the G-actin/F-actin ratio, and impaired myocardin-related transcription factor (MRTF)/serum response factor (SRF)-dependent transcription, which is known to be repressed by increased levels of G-actin. The effect on tubulin acetylation was caused by the almost complete absence of α-tubulin acetyltransferase 1 (α-TAT1) messenger RNA (mRNA). Activation of the MRTF-SRF transcriptional complex restored α-TAT1 mRNA levels and tubulin acetylation. Several functional MRTF-SRF-responsive elements were consistently identified in the α-TAT1 gene. The effect of INF2 silencing on microtubule acetylation was also observed in epithelial ECV304 cells, but not in Jurkat T cells. Therefore, the actin-MRTF-SRF circuit controls α-TAT1 transcription. INF2 regulates the circuit, and hence microtubule acetylation, in cell types where it has a prominent role in actin polymerization.
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an mdia1 INF2 formin activation cascade facilitated by iqgap1 regulates stable microtubules in migrating cells
Molecular Biology of the Cell, 2016Co-Authors: Francesca Bartolini, Nagendran Ramalingam, Laura Andresdelgado, Sara Nik, Leonor Kremer, Miguel A Alonso, Gregg G GundersenAbstract:Multiple formins regulate microtubule (MT) arrays, but whether they function individually or in a common pathway is unknown. Lysophosphatidic acid (LPA) stimulates the formation of stabilized detyrosinated MTs (Glu MTs) in NIH3T3 fibroblasts through RhoA and the formin mDia1. Here we show that another formin, INF2, is necessary for mDia1-mediated induction of Glu MTs and regulation of MT dynamics and that mDia1 can be bypassed by activating INF2. INF2 localized to MTs after LPA treatment in an mDia1-dependent manner, suggesting that mDia1 regulates INF2. Mutants of either formin that disrupt their interaction failed to rescue MT stability in cells depleted of the respective formin, and the mDia1-interacting protein IQGAP1 regulated INF2’s localization to MTs and the induction of Glu MTs by either formin. The N-terminus of IQGAP1 associated with the C-terminus of INF2 directly, suggesting the possibility of a tripartite complex stimulated by LPA. Supporting this, the interaction of mDia1 and INF2 was induced by LPA and dependent on IQGAP1. Our data highlight a unique mechanism of formin action in which mDia1 and INF2 function in series to stabilize MTs and point to IQGAP1 as a scaffold that facilitates the activation of one formin by another.
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Formin INF2 regulates MAL-mediated transport of Lck to the plasma membrane of human T lymphocytes.
Blood, 2010Co-Authors: Laura Andrés-delgado, Ricardo Madrid, Olga M. Antón, Jennifer A. Byrne, Miguel A AlonsoAbstract:Expression of the src-family kinase lymphocyte-specific protein tyrosine kinase (Lck) at the plasma membrane is essential for it to fulfill its pivotal role in signal transduction in T lymphocytes. MAL, an integral membrane protein expressed in specific types of lymphoma, has been shown to play an important role in targeting Lck to the plasma membrane. Here we report that MAL interacts with Inverted Formin2 (INF2), a formin with the atypical property of promoting not only actin polymerization but also its depolymerization. In Jurkat T cells, INF2 colocalizes with MAL at the cell periphery and pericentriolar endosomes and along microtubules. Videomicroscopic analysis revealed that the MAL vesicles transporting Lck to the plasma membrane move along microtubule tracks. Knockdown of INF2 greatly reduced the formation of MAL transport vesicles and the levels of Lck at the plasma membrane and impaired formation of a normal immunologic synapse. The actin polymerization and depolymerization activities of INF2 were both required for efficient Lck targeting. Cdc42 and Rac1, which bind to INF2, regulate Lck transport in both Jurkat and primary human T cells. Thus, INF2 collaborates with MAL in the formation of specific carriers for targeting Lck to the plasma membrane in a process regulated by Cdc42 and Rac1. (Blood. 2010;116(26): 5919-5929)