Biology subjects

Lappalainen, P.

Publications and source records attributed to Lappalainen, P..

14 recordsLinked to original sources

bioRxiv2026

DAAM1 formin and Ena/VASP proteins assemble functionally distinct actin filaments for focal adhesions

Eukaryotic cells contain multiple biochemically distinct actin filament networks, which enable versatile functions of actin in a range of cellular processes. Yet, the mechanisms by which specific actin filament networks are assembled in a common cytoplasm remain elusive. Here, we investigated how functionally distinct actin nanoscale layers, specified by -actininand tropomyosin isoforms, Tpm1.6 and Tpm3.2, are assembled at focal adhesions. By combining genetic perturbations with mitochondrial-targeting of actin polymerases, we discovered that DAAM1 formin assembles Tpm3.2-actin filaments, whereas Ena/VASP family proteins polymerize -actinin cross-linked actin filament bundles at focal adhesions. Consequently, loss of DAAM1 dampened Tpm3.2 protein levels and impaired focal adhesion disassembly, thus phenocopying Tpm3.2-deficient cells. In contrast, Ena/VASP depletion led to defective focal adhesion maturation and loss of -actinin from focal adhesions. More broadly, our study highlights specific roles for formin and Ena/VASP family proteins in assembling biochemically and functionally distinct linear actin filament arrays in cells.

cell biology↗

bioRxiv2024

Differential interference with actin-binding protein function by acute Cytochalasin B

Dynamic actin filament remodeling is crucial for a plethora of fundamental cell biological processes, ranging from cell division and migration to cell communication, intracellular trafficking or tissue development. Cytochalasin B and -D are fungal secondary metabolites frequently used for interference with such processes. Although generally assumed to block actin filament polymerization at their rapidly growing barbed ends and compete with regulators at these sites, our molecular understanding of their precise effects in dynamic actin structures is scarce. Here we combine live cell imaging and analysis of fluorescent actin-binding protein dynamics with acute treatment of lamellipodia in migrating cells with cytochalasin B. Our results show that in spite of an abrupt halt of lamellipodium protrusion, cytochalasin B affects various actin filament barbed end-binding proteins in a differential fashion. Cytochalasin B enhances instead of diminishes the accumulation of prominent barbed end-binding factors such as Ena/VASP family proteins and heterodimeric capping protein (CP) in the lamellipodium. Similar results were obtained with cytochalasin D. All these effects are highly specific, as cytochalasin-induced VASP accumulation requires the presence of CP, but not vice versa, and coincides with abrogation of both actin and VASP turnover. Cytochalasin B can also increase apparent barbed end interactions with the actin-binding {beta}-tentacle of CP and partially mimic its Arp2/3 complex-promoting activity in the lamellipodium. In conclusion, our results reveal a new spectrum of cytochalasin activities on barbed end-binding factors, with important implications for the interpretation of their effects on dynamic actin structures.

cell biology↗

bioRxiv2023

Specialized actin nanoscale layers control focal adhesion turnover

Focal adhesions (FAs) connect inner workings of the cell to the extracellular matrix to control cell adhesion, migration, and mechanosensing1,2. Previous studies demonstrated that FAs contain three vertical layers, which connect extracellular matrix to the cytoskeleton3,4,5. However, cellular processes rely on precisely-regulated FA turnover, but the molecular machineries that control FA assembly and disassembly have remained elusive. By using super-resolution iPALM microscopy, we identified two unprecedented nanoscale layers within FAs, specified by actin filaments bound to tropomyosin isoforms Tpm1.6 and Tpm3.2. The Tpm1.6-actin filaments beneath the previously identified actin-regulatory layer are critical for adhesion maturation and controlled cell motility, whereas the Tpm3.2-actin filament layer towards the bottom of FA facilitates adhesion disassembly. Mechanistically, Tpm3.2 stabilizes KANK-family proteins at adhesions, and hence targets microtubule plus-ends to FAs to catalyse their disassembly. Loss of Tpm3.2 leads to disorganized microtubule network, abnormally stable FAs, and defects in tail retraction during cell migration. Thus, FAs are composed of at least three distinct actin filament layers, each having specific roles in coupling of adhesion to the cytoskeleton, or in controlling adhesion dynamics. In a broader context, these findings demonstrate how distinct actin filament populations can co-exist and perform specific functions within a defined cellular compartment.

cell biology↗

bioRxiv2023

Unusual actin-binding mechanism and the role of profilin in actin dynamics of trypanosomatid parasites

Diseases caused by Leishmania, and Trypanosoma parasites, such as leishmaniasis and African sleeping sickness, are a major health problem in tropical countries. Due to their complex life cycle involving both vertebrate and insect hosts, and > 1 billion years of evolutionarily distance, the cell biology of these trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is highly divergent when compared to the other eukaryotes. To understand how actin dynamics are regulated in trypanosomatid parasites, we focused on a central actin-binding protein profilin. Co-crystal structure of Leishmania major actin in complex with L. major profilin revealed that, although the overall folds of actin and profilin are conserved in eukaryotes, Leishmania profilin contains a unique -helical insertion, which interacts with the target binding cleft of actin monomer. This insertion is conserved across the Trypanosomatidae family, and is strikingly similar to the structure of WH2 domain, a small actin-binding motif found in many other cytoskeletal regulators. We demonstrate that the WH2-like motif contributes to actin monomer-binding and enhances the actin nucleotide exchange activity of Leishmania profilin. Surprisingly, unlike other profilins characterized so far, Leishmania profilin inhibited formin-catalyzed actin filament assembly in a mechanism that is dependent on the presence of the WH2-like motif. By generating profilin knockout and knockin Leishmania mexicana strains, we show that profilin is important for efficient endocytic sorting in parasites, and that the ability to bind actin monomers and proline-rich proteins, as well as the presence of a functional WH2-like motif, are important for the in vivo function of Leishmania profilin. Collectively, this study uncovers the molecular principles by which actin dynamics are regulated by profilin in trypanosomatids. Moreover, the unusual actin-binding mechanism of profilin identified here could be applied for designing inhibitors against pathogenic trypanosomatid parasites. AUTHOR SUMMARYLeishmania and Trypanosoma parasites are a major health problem as they cause various diseases in humans and other vertebrates. Currently, there are no specific drugs to treat the diseases caused by these trypanosomatid parasites. Similar to all other eukaryotes, trypanosomatid parasites have an actin cytoskeleton, which is essential for the viability of parasites. Interestingly, both actin and actin-regulatory machineries of these parasites are highly divergent from the ones of animals, making them possible drug targets to treat diseases caused by these parasites. To uncover how the actin cytoskeleton of trypanosomatid parasites is regulated, we focused on a central actin-binding protein, profilin. Importantly, our experiments revealed that trypanosomatid profilins interact with actin through a different structural mechanism as compared to animal profilins, and have specific effects on the assembly of actin filaments. Our genetic studies demonstrate that these specific features of trypanosomatid profilin are also critical for the proper function on this protein in parasites. Our study provides new insight into the cell biology of trypanosomatid parasites. We also envision that the structural and functional differences between trypanosomatid and human profilins can be applied for developing compounds for selectively neutralizing Leishmania and Trypanosoma parasites.

biochemistry↗

bioRxiv2022

SARS-CoV-2 infected cells sprout actin-rich filopodia that facilitate viral invasion

Emerging COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a great threat to human health and economics. Although SARS-CoV-2 entry mechanism has been explored, little is known about how SARS-CoV-2 regulates the host cell remodeling to facilitate virus invasion process. Here we unveil that SARS-CoV-2 boosts and repurposes filopodia for entry to the target cells. Using SARS-CoV-2 virus-like particle (VLP), real-time live-cell imaging and simulation of active gel model, we reveal that VLP-induced Cdc42 activation leads to the formation of filopodia, which reinforce the viral entry to host cells. By single-particle tracking and sparse deconvolution algorithm, we uncover that VLP particles utilize filopodia to reach the entry site in two patterns, surfing and grabbing, which are more efficient and faster than entry via flat plasma membrane regions. Furthermore, the entry process via filopodia is dependent on the actin cytoskeleton and actin-associated proteins fascin, formin, and Arp2/3. Importantly, either inhibition the actin cross-linking protein fascin or the active level of Cdc42 could significantly hinders both the VLP and the authentic SARS-CoV-2 entry. Together, our results highlight that the spatial-temporal regulation of the actin cytoskeleton by SARS-CoV-2 infection makes filopodia as a highway for virus entry, which emerges as an antiviral target. Significance StatementRevealing the mechanism of SARS-CoV-2 invasion is of great significance to explain its high pathogenic and rapid transmission in the world. We discovered a previously unknown route of SARS-CoV-2 entry. SARS-CoV-2 virus-like particles boost cellular filopodia formation by activating Cdc42. Using state-of-art-technology, we spatial-temporally described how virus utilize filopodia to enter the target cell in two modes: surfing and grabbing. Filopodia can directly transport the virus to endocytic hot spots to avoid the virus from disorderly searching on the plasma membrane. Our study complements current knowledge of SARS-CoV-2 that filopodia and its components not only play an important role in virus release and cell-cell transmission, but also in the entry process, and provides several potential therapeutic targets for SARS-CoV-2. HighlightsO_LISARS-CoV-2 VLP infection promotes filopodia formation by activating Cdc42 C_LIO_LISARS-CoV-2 VLP utilizes filopodia to enter target cell via two modes, surfing and grabbing C_LIO_LIFilopodia disruption compromises the invasion of both VLP and authentic SARS-CoV-2 C_LI

cell biology↗

bioRxiv2022

Recycling limits the lifetime of actin turnover

Intracellular organization is largely mediated by the actin turnover. Cellular actin networks consume matter and energy to sustain their dynamics, while maintaining their appearance. This behavior, called dynamic steady state, enables cells to sense and adapt to their environment. However, how structural stability can be maintained during the constant turnover of a limited actin monomer pool is poorly understood. To answer this question, we developed an experimental system using actin bead motility in a compartment with a limited amount of monomer. We used the speed and the size of the actin comet tails to evaluate the systems monomer consumption and its lifetime. We established the relative contribution of actin assembly, disassembly and recycling for a bead movement over tens of hours. Recycling mediated by cyclase-associated proteins is the key step in allowing the reuse of monomers for multiple assembly cycles. Energy supply and protein aging are also factors that limit the lifetime of actin turnover. This work reveals the balancing mechanism for long-term network assembly with a limited amount of building blocks.

cell biology↗

bioRxiv2022

Structural basis underlying specific biochemical activities of non-muscle tropomyosin isoforms

The actin cytoskeleton is critical for cell migration, morphogenesis, endocytosis, organelle dynamics, and cytokinesis. To support diverse cellular processes, actin filaments form a variety of structures with specific architectures and dynamic properties. Key proteins specifying actin filaments are tropomyosins. Non-muscle cells express several functionally non-redundant tropomyosin isoforms, which differentially control the interactions of other proteins, including myosins and ADF/cofilin, with actin filaments. However, the underlying molecular mechanisms have remained elusive. By determining the cryogenic electron microscopy structures of actin filaments decorated by two functionally distinct non-muscle tropomyosin isoforms, Tpm1.6 and Tpm3.2, we reveal that actin filament conformation remains unaffected upon binding. However, Tpm1.6 and Tpm3.2 follow different paths along the major groove of the actin filament, providing an explanation for their incapability to co-polymerize on actin filaments. The structures and biochemical work also elucidate the molecular basis underlying specific roles of Tpm1.6 and Tpm3.2 in myosin II activation and protecting actin filaments from ADF/cofilin-catalysed severing.

biochemistry↗

bioRxiv2022

Activated I-BAR IRSp53 clustering controls the formation of VASP-actin-based membrane protrusions

Filopodia are actin-rich membrane protrusions essential for cell morphogenesis, motility, and cancer invasion. How cells control filopodia initiation on the plasma membrane remains elusive. We performed experiments in cellulo, in vitro and in silico to unravel the mechanism of filopodia initiation driven by the membrane curvature sensor IRSp53. We showed that full-length IRSp53 self-assembles into clusters on membranes depending on PIP2. Using well-controlled in vitro reconstitution systems, we demonstrated that IRSp53 clusters recruit the actin polymerase VASP to assemble actin filaments locally on membranes, leading to the generation of actin-filled membrane protrusions reminiscent of filopodia. By pulling membrane nanotubes from live cells, we observed that IRSp53 can only be enriched and trigger actin assembly in nanotubes at highly dynamic membrane regions. Our work supports a regulation mechanism of IRSp53 in its attributes of curvature sensation and partner recruitment to ensure a precise spatial-temporal control of filopodia initiation.

biophysics↗

bioRxiv2021

A myosin chaperone, UNC-45A, is a novel regulator of intestinal epithelial barrier integrity and repair

The actomyosin cytoskeleton serves as a key regulator of the integrity and remodeling of epithelial barriers by controlling assembly and functions of intercellular junctions and cell-matrix adhesions. While biochemical mechanisms that regulate activity of non-muscle myosin II (NM-II) in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. UNC-45A is a cytoskeletal chaperone that is essential for proper folding of NM II heavy chains and myofilament assembly. We found abundant expression of UNC-45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein levels of UNC-45A were decreased in colonic epithelium of patients with ulcerative colitis. CRISPR/Cas9-mediated knock-out of UNC-45A in HT-29 and SK-CO15 IEC disrupted epithelial barrier integrity, impaired assembly of epithelial adherence and tight junctions and attenuated cell migration. Consistently, decreased UNC-45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti-migratory effects of UNC-45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC-45A also decreased contractile forces at epithelial junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC-45A in controlling IEC junctions and motility. Our findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.

cell biology↗

bioRxiv2021

Full assembly of HIV-1 particles requires assistance of the membrane curvature factor IRSp53.

During HIV-1 particle formation, the requisite plasma membrane curvature is thought to be solely driven by the retroviral Gag protein. Here, we reveal that the cellular I-BAR protein IRSp53 is required for the progression of HIV-1 membrane curvature to complete particle assembly. Partial gene editing of IRSp53 induces a decrease in viral particle production and a viral bud arrest at half completion. Single molecule localization microscopy at the cell plasma membrane shows a preferential localization of IRSp53 around HIV-1 Gag assembly sites. In addition, we observe the presence of IRSp53 in purified HIV-1 particles. Finally, HIV-1 Gag protein localizes preferentially to IRSp53 I-BAR domain induced curved membranes on giant unilamellar vesicles. Overall, our data reveal a strong interplay between IRSp53 I-BAR and Gag at membranes during virus assembly. This highlights IRSp53 as a crucial host factor in HIV-1 membrane curvature and its requirement for full HIV-1 particle assembly.

biophysics↗

bioRxiv2020

Conformational dynamics regulate SHANK3 actin and Rap1 binding

Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology and specific biological function of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators and small GTPases. SHANK3 is a multifunctional scaffold protein, interacting with several actin-binding proteins, and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its N-terminal SPN domain. Here, we demonstrate that SHANK3 interacts directly with actin using its SPN domain. Actin binding can be inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3 and loss of SHANK3-actin binding augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for filopodia formation in cancer cells, dendritic spine morphology in neurons and autism-linked phenotypes in vivo.

cell biology↗

bioRxiv2020

Generation of stress fibers through myosin-driven re-organization of the actin cortex

Contractile actomyosin bundles, stress fibers, govern key cellular processes including migration, adhesion, and mechanosensing. Stress fibers are thus critical for developmental morphogenesis. The most prominent actomyosin bundles, ventral stress fibers, are generated through coalescence of pre-existing stress fiber precursors. However, whether stress fibers can assemble through other mechanisms has remained elusive. We report that stress fibers can also form without requirement of pre-existing actomyosin bundles. These structures, which we named cortical stress fibers, are embedded in the cell cortex and assemble preferentially underneath the nucleus. In this process, non-muscle myosin II pulses orchestrate the reorganization of cortical actin meshwork into regular bundles, which promote reinforcement of nascent focal adhesions, and subsequent stabilization of the cortical stress fibers. These results identify a new mechanism by which stress fibers can be generated de novo from the actin cortex, and establish role for stochastic myosin pulses in the assembly of functional actomyosin bundles.

cell biology↗

bioRxiv2019

Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

Coordinated polymerization of actin filaments provides force for cell migration, morphogenesis, and endocytosis. Capping Protein (CP) is central regulator of actin dynamics in all eukaryotes. It binds actin filament (F-actin) barbed ends with high affinity and slow dissociation kinetics to prevent filament polymerization and depolymerization. In cells, however, CP displays remarkably rapid dynamics within F-actin networks, but the underlying mechanism has remained enigmatic. We report that a conserved cytoskeletal regulator, twinfilin, is responsible for CPs rapid dynamics and specific localization in cells. Depletion of twinfilin led to stable association of CP with cellular F-actin arrays and its treadmilling throughout leading-edge lamellipodium. These were accompanied by diminished F-actin disassembly rates. In vitro single filament imaging approaches revealed that twinfilin directly promotes dissociation of CP from filament barbed ends, while allowing subsequent filament depolymerization. These results uncover an evolutionary conserved bipartite mechanism that controls how actin cytoskeleton-mediated forces are generated in cells.

cell biology↗

bioRxiv2018

Ezrin enrichment on curved membranes requires a specific conformation or interaction with a curvature-sensitive partner

One challenge in current cell biology is to decipher the biophysical mechanisms governing protein enrichment on curved membranes and the resulting membrane deformation. The ERM protein ezrin is abundant and associated with cellular membranes that are flat or with positive or negative curvatures. Using in vitro and cell biology approaches, we assess mechanisms of ezrins enrichment on curved membranes. We evidence that ezrin (ezrinWT) and its phosphomimetic mutant T567D (ezrinTD) do not deform membranes but self-assemble anti-parallelly, zipping adjacent membranes. EzrinTDs specific conformation reduces intermolecular ezrin interactions, allows binding to actin filaments, and promotes ezrin binding to positively curved membranes. While neither ezrinTD nor ezrinWT senses negative membrane curvature alone, we demonstrate that interacting with curvature sensors I-BAR-domain proteins facilitates ezrin enrichment in negatively curved membrane protrusions. Overall, our work reveals new mechanisms, specific conformation or binding to a curvature sensor partner, for targeting curvature insensitive proteins to curved membranes.

biophysics↗