In mouse brain profilin I and profilin II associate with regulators of the endocytic pathway and actin assembly.

Profilins are thought to be essential for regulation of actin assembly. However, the functions of profilins in mammalian tissues are not well understood. In mice profilin I is expressed ubiquitously while profilin II is expressed at high levels only in brain. In extracts from mouse brain, profilin I and profilin II can form complexes with regulators of endocytosis, synaptic vesicle recycling and actin assembly. Using mass spectrometry and database searching we characterized a number of ligands for profilin I and profilin II from mouse brain extracts including dynamin I, clathrin, synapsin, Rho-associated coiled-coil kinase, the Rac-associated protein NAP1 and a member of the NSF/sec18 family. In vivo, profilins co-localize with dynamin I and synapsin in axonal and dendritic processes. Our findings strongly suggest that in brain profilin I and profilin II complexes link the actin cytoskeleton and endocytic membrane flow, directing actin and clathrin assembly to distinct membrane domains.     

Actin Depolymerizing Factor Is a Component of Slow Axonal Transport

We examined the low molecular weight proteins transported with actin in the chicken sciatic nerve after injection of [35S]methionine into the lumbar spinal cord. A prominent component of slow axonal transport with apparent molecular mass 19 kDa comigrated on two-dimensional gels with chicken actin depolymerizing factor (ADF), previously shown to be a major actin-binding protein in brain. There was comparatively little radioactivity associated with the actin monomer sequestering proteins, profilin or cofilin, and examination of the rapid component of axonal transport failed to reveal appreciable quantities of actin, ADF, profilin, or cofilin. These results show that both actin and ADF are carried by slow axonal transport and raise the possibility that actin travels within the axon in an unpolymerized form in a complex with ADF.     

Profilin is essential for tip growth in the moss Physcomitrella patens

The actin cytoskeleton is critical for tip growth in plants. Profilin is the main monomer actin binding protein in plant cells. The moss Physcomitrella patens has three profilin genes, which are monophyletic, suggesting a single ancestor for plant profilins. Here, we used RNA interference (RNAi) to determine the loss-of-function phenotype of profilin. Reduction of profilin leads to a complete loss of tip growth and a partial inhibition of cell division, resulting in plants with small rounded cells and fewer cells. We silenced all profilins by targeting their 3' untranslated region sequences, enabling complementation analyses by expression of profilin coding sequences. We show that any moss or a lily (Lilium longiflorum) profilin support tip growth. Profilin with a mutation in its actin binding site is unable to rescue profilin RNAi, while a mutation in the poly-l-proline binding site weakly rescues. We show that moss tip growing cells contain a prominent subapical cortical F-actin structure composed of parallel actin cables. Cells lacking profilin lose this structure; instead, their F-actin is disorganized and forms polarized cortical patches. Plants expressing the actin and poly-l-proline binding mutants exhibited similar F-actin disorganization. These results demonstrate that profilin and its binding to actin are essential for tip growth. Additionally, profilin is not needed for formation of F-actin, but profilin and its interactions with actin and poly-l-proline ligands are required to properly organize F-actin.     

Molecular requirements for actin-based lamella formation in Drosophila S2 cells

Cell migration occurs through the protrusion of the actin-enriched lamella. Here, we investigated the effects of RNAi depletion of approximately 90 proteins implicated in actin function on lamella formation in Drosophila S2 cells. Similar to in vitro reconstitution studies of actin-based Listeria movement, we find that lamellae formation requires a relatively small set of proteins that participate in actin nucleation (Arp2/3 and SCAR), barbed end capping (capping protein), filament depolymerization (cofilin and Aip1), and actin monomer binding (profilin and cyclase-associated protein). Lamellae are initiated by parallel and partially redundant signaling pathways involving Rac GTPases and the adaptor protein Nck, which stimulate SCAR, an Arp2/3 activator. We also show that RNAi of three proteins (kette, Abi, and Sra-1) known to copurify with and inhibit SCAR in vitro leads to SCAR degradation, revealing a novel function of this protein complex in SCAR stability. Our results have identified an essential set of proteins involved in actin dynamics during lamella formation in Drosophila S2 cells.     

Drosophila fragile X mental retardation protein developmentally regulates activity-dependent axon pruning

Fragile X Syndrome (FraX) is a broad-spectrum neurological disorder with symptoms ranging from hyperexcitability to mental retardation and autism. Loss of the fragile X mental retardation 1 (fmr1) gene product, the mRNA-binding translational regulator FMRP, causes structural over-elaboration of dendritic and axonal processes, as well as functional alterations in synaptic plasticity at maturity. It is unclear, however, whether FraX is primarily a disease of development, a disease of plasticity or both: a distinction that is vital for engineering intervention strategies. To address this crucial issue, we have used the Drosophila FraX model to investigate the developmental function of Drosophila FMRP (dFMRP). dFMRP expression and regulation of chickadee/profilin coincides with a transient window of late brain development. During this time, dFMRP is positively regulated by sensory input activity, and is required to limit axon growth and for efficient activity-dependent pruning of axon branches in the Mushroom Body learning/memory center. These results demonstrate that dFMRP has a primary role in activity-dependent neural circuit refinement during late brain development.     

A balance of capping protein and profilin functions is required to regulate actin polymerization in Drosophila bristle

Profilin is a well-characterized protein known to be important for regulating actin filament assembly. Relatively few studies have addressed how profilin interacts with other actin-binding proteins in vivo to regulate assembly of complex actin structures. To investigate the function of profilin in the context of a differentiating cell, we have studied an instructive genetic interaction between mutations in profilin (chickadee) and capping protein (cpb). Capping protein is the principal protein in cells that caps actin filament barbed ends. When its function is reduced in the Drosophila bristle, F-actin levels increase and the actin cytoskeleton becomes disorganized, causing abnormal bristle morphology. chickadee mutations suppress the abnormal bristle phenotype and associated abnormalities of the actin cytoskeleton seen in cpb mutants. Furthermore, overexpression of profilin in the bristle mimics many features of the cpb loss-of-function phenotype. The interaction between cpb and chickadee suggests that profilin promotes actin assembly in the bristle and that a balance between capping protein and profilin activities is important for the proper regulation of F-actin levels. Furthermore, this balance of activities affects the association of actin structures with the membrane, suggesting a link between actin filament dynamics and localization of actin structures within the cell.     

A role for complexes of survival of motor neurons (SMN) protein with gemins and profilin in neurite-like cytoplasmic extensions of cultured nerve cells

Spinal muscular atrophy (SMA) is caused by reduced levels of SMN (survival of motor neurons protein) and consequent loss of motor neurons. SMN is involved in snRNP transport and nuclear RNA splicing, but axonal transport of SMN has also been shown to occur in motor neurons. SMN also binds to the small actin-binding protein, profilin. We now show that SMN and profilin II co-localise in the cytoplasm of differentiating rat PC12 cells and in neurite-like extensions, especially at their growth cones. Many components of known SMN complexes were also found in these extensions, including gemin2 (SIP-1), gemin6, gemin7 and unrip (unr-interacting protein). Coilin p80 and Sm core protein immunoreactivity, however, were seen only in the nucleus. SMN is known to associate with beta-actin mRNA and specific hnRNPs in axons and in neurite extensions of cultured nerve cells, and SMN also stimulates neurite outgrowth in cultures. Our results are therefore consistent with SMN complexes, rather than SMN alone, being involved in the transport of actin mRNPs along the axon as in the transport of snRNPs into the nucleus by similar SMN complexes. Antisense knockdown of profilin I and II isoforms inhibited neurite outgrowth of PC12 cells and caused accumulation of SMN and its associated proteins in cytoplasmic aggregates. BIAcore studies demonstrated a high affinity interaction of SMN with profilin IIa, the isoform present in developing neurons. Pathogenic missense mutations in SMN, or deletion of exons 5 and 7, prevented this interaction. The interaction is functional in that SMN can modulate actin polymerisation in vitro by reducing the inhibitory effect of profilin IIa. This suggests that reduced SMN in SMA might cause axonal pathfinding defects by disturbing the normal regulation of microfilament growth by profilins.     

In vivo importance of actin nucleotide exchange catalyzed by profilin

The actin monomer-binding protein, profilin, influences the dynamics of actin filaments in vitro by suppressing nucleation, enhancing nucleotide exchange on actin, and promoting barbed-end assembly. Profilin may also link signaling pathways to actin cytoskeleton organization by binding to the phosphoinositide PIP(2) and to polyproline stretches on several proteins. Although activities of profilin have been studied extensively in vitro, the significance of each of these activities in vivo needs to be tested. To study profilin function, we extensively mutagenized the Saccharomyces cerevisiae profilin gene (PFY1) and examined the consequences of specific point mutations on growth and actin organization. The actin-binding region of profilin was shown to be critical in vivo. act1-157, an actin mutant with an increased intrinsic rate of nucleotide exchange, suppressed defects in actin organization, cell growth, and fluid-phase endocytosis of pfy1-4, a profilin mutant defective in actin binding. In reactions containing actin, profilin, and cofilin, profilin was required for fast rates of actin filament turnover. However, Act1-157p circumvented the requirement for profilin. Based on the results of these studies, we conclude that in living cells profilin promotes rapid actin dynamics by regenerating ATP actin from ADP actin-cofilin generated during filament disassembly.     

no-bridge and linotte act jointly at the interhemispheric junction to build up the adult central brain of Drosophila melanogaster

The Drosophila transmembrane protein Linotte (Lio) is expressed in a glial transcient interhemispheric fibrous ring (TIFR), which was hypothesised to serve as scaffold for adult brain formation during metamorphosis. We isolated TIFR specific enhancers from the lio locus and showed that only four interhemispheric cells give rise to this complex fibrous structure. We showed that lio controls the TIFR differentiation, and confirmed the major role played by this structure in central brain metamorphosis since its destruction by apoptosis led to a pronounced adult phenotype, which included defects of lio and no-bridge (nob) mutants. lio interhemispheric expression was specifically affected in a nob(1) context, confirming that nob plays a key role in adult brain development via the TIFR.     

Exportin 6: a novel nuclear export receptor that is specific for profilin.actin complexes

Active macromolecular transport between the nucleus and cytoplasm proceeds through nuclear pore complexes and is mostly mediated by transport receptors of the importin beta-superfamily. Here we identify exportin 6 (Exp6) as a novel family member from higher eukaryotes and show that it mediates nuclear export of profilin.actin complexes. Exp6 appears to contact primarily actin, but the interaction is greatly enhanced by the presence of profilin. Profilin thus functions not only as the nucleotide exchange factor for actin, but can also be regarded as a cofactor of actin export and hence as a suppressor of actin polymerization in the nucleus. Even though human and Drosophila Exp6 share only approximately 20% identical amino acid residues, their function in profilin.actin export is conserved. A knock-down of Drosophila Exp6 by RNA interference abolishes nuclear exclusion of actin and results in the appearance of nuclear actin paracrystals. In contrast to a previous report, we found no indications of a major and direct role for CRM1 in actin export from mammalian or insect nuclei.     

Early development of the Drosophila mushroom bodies, brain centres for associative learning and memory

We have studied the formation of Drosophila mushroom bodies using enhancer detector techniques to visualize specific components of these complex intrinsic brain structures. During embryogenesis, neuronal proliferation begins in four mushroom body neuroblasts and the major axonal pathways of the mushroom bodies are pioneered. During larval development, neuronal proliferation continues and further axonal projections in the pedunculus and lobes are formed in a highly structured manner characterized by spatial heterogeneity of reporter gene expression. Enhancer detector analysis identifies many genomic locations that are specifically activated in mushroom body intrinsic neurons (Kenyon cells) during the transition from embryonic to postembryonic development and during metamorphosis.     

Amyloid precursor protein promotes post-developmental neurite arborization in the Drosophila brain

The mechanisms regulating the outgrowth of neurites during development, as well as after injury, are key to the understanding of the wiring and functioning of the brain under normal and pathological conditions. The amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease (AD). However, its physiological role in the central nervous system is not known. Many physical interactions between APP and intracellular signalling molecules have been described, but their functional relevance remains unclear. We show here that human APP and Drosophila APP-Like (APPL) can induce postdevelopmental axonal arborization, which depends critically on a conserved motif in the C-terminus and requires interaction with the Abelson (Abl) tyrosine kinase. Brain injury induces APPL upregulation in Drosophila neurons, correlating with increased post-traumatic mortality in appl(d) mutant flies. Finally, we also found interactions between APP and the JNK stress kinase cascade. Our findings suggest a role for APP in axonal outgrowth after traumatic brain injury.     

Respective roles of the DRL receptor and its ligand WNT5 in Drosophila mushroom body development

In recent decades, Drosophila mushroom bodies (MBs) have become a powerful model for elucidating the molecular mechanisms underlying brain development and function. We have previously characterized the derailed (drl; also known as linotte) receptor tyrosine kinase as an essential component of adult MB development. Here we show, using MARCM clones, a non-cell-autonomous requirement for the DRL receptor in MB development. This result is in accordance with the pattern of DRL expression, which occurs throughout development close to, but not inside, MB cells. While DRL expression can be detected within both interhemispheric glial and commissural neuronal cells, rescue of the drl MB defects appears to involve the latter cellular type. The WNT5 protein has been shown to act as a repulsive ligand for the DRL receptor in the embryonic central nervous system. We show here that WNT5 is required intrinsically within MB neurons for proper MB axonal growth and probably interacts with the extrinsic DRL receptor in order to stop axonal growth. We therefore propose that the neuronal requirement for both proteins defines an interacting network acting during MB development.     

Drosophila Ten-m and filamin affect motor neuron growth cone guidance

The Drosophila Ten-m (also called Tenascin-major, or odd Oz (odz)) gene has been associated with a pair-rule phenotype. We identified and characterized new alleles of Drosophila Ten-m to establish that this gene is not responsible for segmentation defects but rather causes defects in motor neuron axon routing. In Ten-m mutants the inter-segmental nerve (ISN) often crosses segment boundaries and fasciculates with the ISN in the adjacent segment. Ten-m is expressed in the central nervous system and epidermal stripes during the stages when the growth cones of the neurons that form the ISN navigate to their targets. Over-expression of Ten-m in epidermal cells also leads to ISN misrouting. We also found that Filamin, an actin binding protein, physically interacts with the Ten-m protein. Mutations in cheerio, which encodes Filamin, cause defects in motor neuron axon routing like those of Ten-m. During embryonic development, the expression of Filamin and Ten-m partially overlap in ectodermal cells. These results suggest that Ten-m and Filamin in epidermal cells might together influence growth cone progression.     

The Drosophila fragile X mental retardation protein controls actin dynamics by directly regulating profilin in the brain

Loss of Fragile X mental retardation protein (FMRP) function causes the highly prevalent Fragile X syndrome [1 and 2]. Identifying targets for the RNA binding FMRP is a major challenge and an important goal of research into the pathology of the disease. Perturbations in neuronal development and circadian behavior are seen in Drosophila dfmr1 mutants. Here we show that regulation of the actin cytoskeleton is under dFMRP control. dFMRP binds the mRNA of the Drosophila profilin homolog and negatively regulates Profilin protein expression. An increase in Profilin mimics the phenotype of dfmr1 mutants. Conversely, decreasing Profilin levels suppresses dfmr1 phenotypes. These data place a new emphasis on actin misregulation as a major problem in fmr1 mutant neurons.     

act up controls actin polymerization to alter cell shape and restrict Hedgehog signaling in the Drosophila eye disc

Cells in the morphogenetic furrow of the Drosophila eye disc undergo a striking shape change immediately prior to their neuronal differentiation. We have isolated mutations in a novel gene, act up (acu), that is required for this shape change. acu encodes a homolog of yeast cyclase-associated protein, which sequesters monomeric actin; we show that acu is required to prevent actin filament polymerization in the eye disc. In contrast, profilin promotes actin filament polymerization, acting epistatically to acu. However, both acu and profilin are required to prevent premature Hedgehog-induced photoreceptor differentiation ahead of the morphogenetic furrow. These findings suggest that dynamic changes in actin filaments alter cell shape to control the movement of signals that coordinate a wave of differentiation.     

Microinjection of profilins from different sources into the green algaMicrasterias causes transient inhibition of cell growth

Summary Recombinant profilins from different sources (Betula verrucosa, Schizosaccharomyces pombe, Acanthamoeba castellani, or man) cause marked effects on cell growth and morphogenesis when microinjected into growing cells of the green algaMicrasterias denticulata. Whereas control injections with -lactoglobulin only result in a slight delay of cell growth, when profilin is injected cell differentiation ceases and only resumes about 1 to 2 h after the injection, depending on the dose. The resulting cell does not show any malformations, but is reduced in size and retarded in differentiation compared to controls. As a consequence of the profilin microinjection the pattern of cytoplasmic streaming and cytoplasmic structure are also altered. Gelsolin, injected for comparison, leads to minor retardation of cell development but produces less marked effects than profilin. Microinjection of fluorescently labeled profilin shows even distribution throughout the cytoplasm and more intense fluorescence in the nucleus. Electron microscopical investigations of cells fixed immediately after profilin injection show a normal distribution of dictyosomes, ER cisternae, microtubules, and secretory vesicles compared to noninjected controls at the same developmental stage. Our results indicate that disturbance of the natural actin turnover by the injection of actin-binding proteins strongly affects development ofMicrasterias, corroborating a key role of actin in the morphogenetic process.     

Slow Axonal Transport of Soluble Actin with Actin Depolymerizing Factor, Cofilin, and Profilin Suggests Actin Moves in an Unassembled Form

Abstract: We examined the axonal transport of actin and its monomer binding proteins, actin depolymerizing factor, cofilin, and profilin, in the chicken sciatic nerve following injection of [³⁵S]methionine into the lumbar spinal cord. At intervals up to 20 days after injection, nerves were cut into 1-cm segments and separated into Triton X-100-soluble and particulate fractions. Actin and its binding proteins were then isolated by affinity chromatography on DNase I-Sepharose and by one- and two-dimensional polyacrylamide gel electrophoresis. Fluorographic analysis showed that the specific activity of soluble actin was two to three times that of its particulate form and that soluble actin, cofilin, actin depolymerizing factor, and profilin were transported at similar rates in slow component b of axonal flow. Our data strongly support the view that the mobile form of actin in slow transport is soluble and that a substantial amount of this actin may travel as a complex with actin depolymerizing factor, cofilin, and profilin. Along labeled nerves the specific activity of the unphosphorylated form of actin depolymerizing factor, which binds actin, was not significantly different from that of its "inactive" phosphorylated form. This constancy in specific activity suggests that continuous inactivation and reactivation of actin depolymerizing factor occur during transport, which could contribute to the exchange of soluble actin with the filamentous actin pool.     

The actin released from profilin--actin complexes is insufficient to account for the increase in F-actin in chemoattractant-stimulated polymorphonuclear leukocytes

Chemoattractant stimulation of polymorphonuclear leukocytes is associated with a nearly two-fold rise in actin filament content. We examined the role of the actin monomer sequestering protein, profilin, in the regulation of PMN actin filament assembly during chemoattractant stimulation using a Triton extraction method. Poly-L-proline-conjugated Sepharose beads were used to assess the relative concentration of actin bound to profilin with high enough affinity to withstand dilution (profilin-actin complex) and DNase I-conjugated beads to measure the relative concentration of actin in the Triton-soluble fraction not bound to profilin. Actin associated with the Triton-insoluble fraction (F-actin) was also measured. In unstimulated PMN, the relative concentration of actin bound to profilin was maximum. After FMLP stimulation, profilin released actin monomers within 10 s, with the profilin-actin complex concentration reaching a nadir by 40 s and remaining low as long as the cells were exposed to chemoattractant (up to 30 min). If FMLP was dissociated from PMN membrane receptors using t-BOC, actin reassociated with profilin within 20 s. Quantitative analysis of these reactions, however, revealed that profilin release of and rebinding to actin could account for only a small percentage of the total change in F-actin content. Determination of the total profilin and actin concentrations in PMN revealed that the molar ratio of profilin to actin was 1 to 5.2. When purified actin was polymerized in PMN Triton extract containing EGTA, removal of profilin from the extract minimally affected (12% reduction) the high apparent critical concentration at which actin began to assemble. Although profilin released actin at the appropriate time to stimulate actin assembly during exposure to chemoattractants, the concentration of profilin in PMN was insufficient to explain the high unpolymerized actin content in unstimulated PMN and the quantity of actin released from profilin too small to account for the large shifts from unpolymerized to polymerized actin associated with maximal chemoattractant stimulation.