Cell Migration: Pump up the volume
An influx of water molecules can help immune cells called neutrophils to move to where they are needed in the body.
- Institute for NanoBioTechnology and Department of Mechanical Engineering, Johns Hopkins University, United States
Cell migration is essential for most processes in the body, such as embryonic development, immune responses or wound repair. The ability of cells to migrate is a complex phenomenon that depends on many factors, including the polymerization of actin filaments. For a cell to move, actin filaments and other proteins in the cytoskeleton actively polymerize against the cell membrane to generate protrusions and push the cell surface forward.
In recent years, however, it has become evident that ion and water flow through the cell membrane might also be involved in cell movement (Stroka et al., 2014; Li et al., 2020). A polarized distribution of ion channels and transporters can generate a small osmolarity gradient in the cytoplasm, and the resulting influx of ions and water molecules can cause the cell to swell at its leading edge and to shrink at its trailing edge, thereby enabling it to move. However, the mechanisms underlying these processes are not fully understood.
Now, in eLife, Tamas Nagy, Evelyn Strickland and Orion Weiner at the University of California San Francisco report that innate immune cells known as neutrophils rely on water flux to help them move quickly to the sites of infection or injury (Nagy et al., 2024). It is well known that neutrophils use actin to relocate, but it has also been shown that they can increase the influx of water into the cell – and consequently their volume and motility – in response to molecules known as chemoattractants (Weiner et al., 1999).
To better understand the impact of water and ions on cell movement, Nagy et al. used a technique, called Fluorescence Exclusion Method, which can track changes in the volumes of single cells (Cadart et al., 2017). This revealed that when the neutrophils were exposed to chemoattractants, the cells started to swell and became mobile.
Building on these findings, Nagy et al. sought to identify the molecules that regulate water-driven migration in neutrophils: this was a challenging task because numerous ion transporters and regulatory proteins are involved in the process (Hoffmann et al., 2009). To navigate this complexity, they combined genome-wide CRISPR/Cas9 knockout screening with an approach that separated cells according to their buoyant density (Shalem et al., 2014). In brief, they generated single-gene knockouts for every gene in the genome and identified which knockouts swelled when exposed to chemoattractants, and which did not.
Many of the genes they identified as being involved in cell swelling and migration were already known, such as the genes encoding the ion transporters NHE1 and AE2 (Li et al., 2021). However, a gene called PI3Kγ – primarily known for its roles in cell growth and cell phenotype specification – also emerged as a candidate (Mendoza et al., 2011; Madsen, 2020). Nagy et al. then performed further experiments to confirm that these regulators were responsible for the positive correlation between the changes in cell volume and migration velocity. Tests involving hypo-osmotic shocks provided evidence of the relationship between NHE1-driven water flux and cell motility. In summary, Nagy et al. demonstrated that cell swelling is both necessary and sufficient for neutrophils to move following stimulation with chemoattractants. It also complements cytoskeletal rearrangements to enhance migration speed.
The exciting findings by Nagy et al. usher in a new era of exploration that intersects cytoskeletal dynamics and cell electrophysiology. Intracellular electrophysiological homeostasis – the balance between ions, proteins and water – is maintained by a complex system involving numerous ion transporters and the actin cytoskeleton.
Conversely, the intracellular ionic environment can influence cytoskeletal activity and force generation (de Boer et al., 2023; Webb et al., 2011). Previous studies have shown that molecular interactions between actin, NHE1 and Akt (which is a target of PI3K) regulate actin organization, intracellular pH and cell migration, while recent work suggests that non-cancerous cells often use actin-NHE1 crosstalk to mediate mechanosensitive adaptations to environmental stimuli (Denker et al., 2000; Denker and Barber, 2002; Meima et al., 2009; Ni et al., 2024).
Collectively, these studies highlight a deeply interconnected system where PI3K and Akt form a hub that potentially links the mechanical ‘players’ in the cell (such as F-actin and myosin II) with the electrophysiological players (such as NHE1)(De Belly et al., 2023), thereby regulating cell migration, mechanosensation and growth.
Given the complicated nature of this mechano-electrophysiological system, comprehensive, high-throughput methods (such as genome-wide knockout screening) are highly valuable. Mathematical models based on such large-scale data will also be instrumental in understanding the underlying interactions. For example, large-scale genomic datasets have revealed the importance of ion transporters in cancer cells, with key elements once again being ion transporters in the NHE and AE families (Shorthouse et al., 2018). It is likely that the system governing ionic and water content regulation, cell migration and metabolism forms the basis of essential biological processes such as growth and morphogenesis, and that alterations in the system could be the origin of many diseases.
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© 2024, Ni and Sun
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Cell Migration: Pump up the volume
eLife 13:e100032.
https://doi.org/10.7554/eLife.100032
Further reading
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- Cell Biology
Distal appendages are nine-fold symmetric blade-like structures attached to the distal end of the mother centriole. These structures are critical for formation of the primary cilium, by regulating at least four critical steps: ciliary vesicle recruitment, recruitment and initiation of intraflagellar transport (IFT), and removal of CP110. While specific proteins that localize to the distal appendages have been identified, how exactly each protein functions to achieve the multiple roles of the distal appendages is poorly understood. Here we comprehensively analyze known and newly discovered distal appendage proteins (CEP83, SCLT1, CEP164, TTBK2, FBF1, CEP89, KIZ, ANKRD26, PIDD1, LRRC45, NCS1, CEP15) for their precise localization, order of recruitment, and their roles in each step of cilia formation. Using CRISPR-Cas9 knockouts, we show that the order of the recruitment of the distal appendage proteins is highly interconnected and a more complex hierarchy. Our analysis highlights two protein modules, CEP83-SCLT1 and CEP164-TTBK2, as critical for structural assembly of distal appendages. Functional assays revealed that CEP89 selectively functions in RAB34+ ciliary vesicle recruitment, while deletion of the integral components, CEP83-SCLT1-CEP164-TTBK2, severely compromised all four steps of cilium formation. Collectively, our analyses provide a more comprehensive view of the organization and the function of the distal appendage, paving the way for molecular understanding of ciliary assembly.
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- Cell Biology
The primary cilium is a microtubule-based organelle that cycles through assembly and disassembly. In many cell types, formation of the cilium is initiated by recruitment of ciliary vesicles to the distal appendage of the mother centriole. However, the distal appendage mechanism that directly captures ciliary vesicles is yet to be identified. In an accompanying paper, we show that the distal appendage protein, CEP89, is important for the ciliary vesicle recruitment, but not for other steps of cilium formation (Tomoharu Kanie, Love, Fisher, Gustavsson, & Jackson, 2023). The lack of a membrane binding motif in CEP89 suggests that it may indirectly recruit ciliary vesicles via another binding partner. Here, we identify Neuronal Calcium Sensor-1 (NCS1) as a stoichiometric interactor of CEP89. NCS1 localizes to the position between CEP89 and a ciliary vesicle marker, RAB34, at the distal appendage. This localization was completely abolished in CEP89 knockouts, suggesting that CEP89 recruits NCS1 to the distal appendage. Similarly to CEP89 knockouts, ciliary vesicle recruitment as well as subsequent cilium formation was perturbed in NCS1 knockout cells. The ability of NCS1 to recruit the ciliary vesicle is dependent on its myristoylation motif and NCS1 knockout cells expressing a myristoylation defective mutant failed to rescue the vesicle recruitment defect despite localizing properly to the centriole. In sum, our analysis reveals the first known mechanism for how the distal appendage recruits the ciliary vesicles.
