Role of septins in animal cell morphogenesis – SEPTIMORF

The methodology of SEPTIMORF relies on combining a reductionist study using cell-free reconstitution with studies in cells. To reconstitute septin organization and function, we observe and analyze purified septin hexamers or octamers, alone, in the presence of actin filaments, microtubules, or biomimetic membranes. To examine how septins interact with the different components, we use a variety of microscopy techniques that allow us to observe the organization of molecules at the micrometric and nanometric scales. To determine if septins in cells exist as filaments, we developed a protocol that produces fluorescence when specific septin-septin interactions occur. We further developed a mutant that impairs septin polymerization and a mutant that disrupts hexamers while preserving octamers, allowing us to study the respective functions. To study septin-membrane interactions in cells, we used a technique that measures the proximity of septins to membranes with respect to actin. Finally, to identify which part of septins binds microtubules, we pursued a bioinformatics approach for microtubule-binding proteins containing sequences similar to septins, and generated a series of mutants for probing septin-microtubule binding.

SEPTIMORF has led to three major results. First, we showed that the lipid composition of the membrane has a dramatic impact on how septins organize. Second, we succeeded to identify the microtubule-binding domain of septins and also showed that the SEPT9 sequence harboring HNA mutations has a regulatory role in septin-microtubule association, but was not required for this association. We finally showed that all actin filament-associated septins in cells exist in the form of filaments containing exclusively octamers which function to anchor actin to the cell membrane. These results are of broad scientific interest, with impact in multiple fields, from cell and developmental biology to mechanobiology and in research on cancer, neurodegenerative and neuro-muscular diseases.

Considering the ubiquitous expression of septins in human tissues, the involvement of septins in multiple human diseases, including the SEPT9-specific disease Hereditary Neuralgic Amyotrophy (HNA), the highly conserved nature of animal septins and the established role of septins in animal cell division and motility, the results of SEPTIMORF are of broad scientific interest and will have impact in multiple fields, from cell and developmental biology to mechanobiology. Research in septin biology and in broader cytoskeleton biology, but also in cell and tissue morphogenesis will be able to build on our findings in order to explore the molecular basis of septin function in a multicellular context and in the context of pathophysiology. Importantly, SEPTIMORF's results will provide the scientific community with a large range of novel tools for studying septin organization in vitro and in cells: the generation of recombinant human septin octamers, the identification of the MT-binding domain, the development of the split-GFP assay to probe specific septin-septin interactions, and the development of new mutants impairing selectively hexamers while preserving octamers, are examples of such tools that promise to be widely used in multiple contexts. The tools we have developed can be readily adapted for studies in genetically tractable animal model systems, which will help advance septin biology studies in a multicellular context. To contribute to open science and facilitate studies using our new tools, we have deposited all plasmids used in our studies with the nonprofit repository Addgene. It is further important to emphasize that SEPTIMORF showcases the joint efforts of physicists and biologists. The success of our approach will reinforce the potential of multi-scale interdisciplinary approaches for elucidating septin function and more broadly biological function, and will also provide a paradigm for inspiring and training the next generation of scientists with an interdisciplinary education. SEPTIMORF involved precisely early-career scientists, PhD students and postdocs, who greatly benefited from accomplishing their research immersed in such an interdisciplinary envinronment.
Our work has provided answers to fundamental questions, but has also highlighted the importance of working actively towards four main objectives in future studies. First, the identification of the actin-binding domain and of the membrane-binding domain in septins will allow us to probe directly how membrane-septin-actin binding interplays with septin function. Second, the nanometric structure of actin-septin, membrane-septin and MT-septin interactions will be key for us to be able to decipher how all these interactions work together to enable septin function. Finally, studying septins in a multicellular context promises to lead to significant advances in our understanding of septin function in a physiological context.

The work of SEPTIMORF has led to seven publications in international peer-reviewed journals with very high visibility in the cell biology field, including the journals eLife and the Journal of Cell Science. These publications are jointly authored by several members of the consortium reflecting the genuine interdisciplinary character of the project.

Septins are conserved, ubiquitous cytoskeletal proteins that have a central role in cell division, cell motility and animal cell morphogenesis. Septins organize into palindromic protomers (hexamers and octamers), which can polymerize into filaments and bind the plasma membrane, as well as actin filaments and microtubules. Although human septin dysfunction is linked to infertility, neurodegenerative diseases and cancer, the molecular mechanisms underlying the function of human septins are not clear. SEPTIMORF aims at elucidating the function of human septins by using animal cell division as a model morphogenetic process whose success depends on septins. Several different septin protomers interact with membranes, actin and microtubules, yet how these different protomers differentially contribute to function is not known. Our central hypothesis is that the type of septin protomer tunes the ability of septins to polymerize and determines their affinity and specificity for membranes, actin filaments and microtubules, and thereby determines septin function. To decipher the link between human septin organization and function and to tease apart the hierarchy of septin interactions with other cytoskeletal elements, SEPTIMORF uniquely combines bottom-up approaches using purified components with functional studies in dividing cells. Our multi-scale interdisciplinary approach will provide a first of its kind comprehensive understanding of how septins functionally organize in human cells thus providing insights into the role of septins in health and disease.