Biology subjects

Kotila, T.

Publications and source records attributed to Kotila, T..

5 recordsLinked to original sources

bioRxiv2025

Rapid apical actin filament turnover is required for non-centrosomal microtubule organization in epithelium

Epithelial cells rely on the precise coordination of actin and microtubule cytoskeletons to maintain their polarized structure and function. Within polarized epithelial cells, microtubules form longitudinal non-centrosomal arrays essential for organelle positioning and facilitate directional cargo trafficking whereas the apical actin cytoskeleton supports cell shape and tissue architecture. This study investigates the interplay between disrupted apical actin dynamics and microtubule organization in vivo within Drosophila epithelial tissue. Loss of actin turnover promoting Cyclase-Associated Protein (CAP) results in the excessive accumulation of stable actin structures at the apical cortex. We show that accumulated apical actin sterically excludes microtubules and membrane-bound vesicles. Consequently, non-centrosomal microtubule-dependent processes are impaired, leading to nuclear mispositioning, disrupted apical cargo transport, and defective microvilli formation in CAP mutant cells. Our findings highlight that dense apical actin, due to impaired actin turnover catalyzed by CAP, disrupts the apical organization of non-centrosomal microtubules. This suggests that spatially regulated actin filament turnover is important for microtubule organization and sustaining the polarized functions of epithelial tissues.

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

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↗

bioRxiv2018

Revolutionising the design and analysis of protein engineering experiments using fractional factorial design

Protein engineering is one of the foundations of biotechnology, used to increase protein stability, re-assign the catalytic properties of enzymes or increase the interaction affinity between antibody and target. To date, strategies for protein engineering have focussed on systematic, random or computational methods for introducing new mutations. Here, we introduce the statistical approach of fractional factorial design as a convenient and powerful tool for the design and analysis of protein mutations, allowing sampling of a large mutational space whilst minimising the tests to be done. Our test case is the integral membrane protein, Acridine resistance subunit B (AcrB), part of the AcrAB-TolC multi-protein complex, a multi-drug efflux pump of Gram-negative bacteria. E. coli AcrB is naturally histidine-rich, meaning that it is a common contaminant in the purification of recombinantly expressed, histidine-tagged membrane proteins. Coupled with the ability of AcrB to crystallise from picogram quantities causing false positives in 2-D and 3-D crystallisation screening, AcrB contamination represents a significant hindrance to the determination of new membrane protein structures. Here, we demonstrate the use of fractional factorial design for protein engineering, identifying the most important residues involved in the interaction between AcrB and nickel resin. We demonstrate that a combination of spatially close, but sequentially distant histidine residues are important for nickel binding, which were different from those predicted a priori. Fractional factorial methodology has the ability to decrease the time and material costs associated with protein engineering whilst expanding the depth of mutational space explored; a revolutionary concept.\n\nSignificance statementProtein engineering is important for the production of enzymes for bio-manufacturing, stabilised protein for research and production of therapeutic antibodies against human diseases. Here, we introduce a statistical method that can reduce the time and cost required to perform protein engineering. We validate our approach experimentally using the multi-drug efflux pump AcrB, a target for understanding drug-resistance in pathogenic bacteria, but also a persistent contaminant in the purification of membrane proteins from E. coli. This provides a general method for increasing the efficiency of protein engineering.

bioengineering↗