A cytoskeleton symphony: Actin and microtubules in microglia dynamics and aging

Microglia dynamically reorganize their cytoskeleton to perform essential functions such as phagocytosis of toxic protein aggregates, surveillance of the brain parenchyma, and regulation of synaptic plasticity during neuronal activity bursts. Recent studies have shed light on the critical role of the microtubule cytoskeleton in microglial reactivity and function, revealing key regulators like cyclin-dependent kinase 1 and centrosomal nucleation in the remodeling of microtubules in activated microglia. Concurrently, the role of the actin cytoskeleton is also pivotal, particularly in the context of small GTPases like RhoA, Rac1, and Cdc42 and actin-binding molecules such as profilin-1 and cofilin. This article delves into the intricate molecular landscape of actin and microtubules, exploring their synergistic roles in driving microglial cytoskeletal dynamics. We propose a more integrated view of actin and microtubule cooperation, which is fundamental to understanding the functional coherence of the microglial cytoskeleton and its pivotal role in propelling brain homeostasis. Furthermore, we discuss how alterations in microglial cytoskeleton dynamics during aging and in disease states could have far-reaching implications for brain function. By unraveling the complexities of microglia cytoskeletal dynamics, we can deepen our understanding of microglial functional states and their implications in health and disease, offering insights into potential therapeutic interventions for neurologic disorders.


Introduction
The study of microglial reactivity has traditionally focused on the roles of actin and microtubules, viewing them as separate elements with distinct functions.Actin is closely associated with immediate and localized microglial functions, such as engulfing debris, navigating the brain parenchyma, and monitoring synaptic activity.On the other hand, microtubules have been thought to contribute to more prolonged and extensive cellular transformations, such as cell division and intracellular trafficking.However, recent advances in cellular and molecular neuroscience suggest that the dichotomy between actin and microtubules is less clear-cut than previously believed.Instead, these two cytoskeletal components interact in a more integrated and synergistic way, influencing the cell's shape across various microglial functional statessuch as homeostasis, activation, or aging (Fig. 1A).This updated understanding not only bridges the gap between the immediate and prolonged aspects of microglial functionality but also offers novel insights into how these cytoskeletal systems adapt and respond to diverse environmental demands, particularly in the context of aging and disease.

Actin: expanding the classical molecular landscape
Actin, a highly conserved protein found in all eukaryotic cells, plays a fundamental role in various cellular processes due to its dynamic structure and ability to form different types of filaments.In its monomeric form, known as G-actin (globular actin), it has the potential to polymerize into F-actin (filamentous actin), a process crucial for cell motility, shape, and division.Actin polymerization, the assembly of Gactin into F-actin, is a dynamic and tightly regulated process influenced by various actin-binding proteins that control the rate and extent of filament growth and disassembly.This dynamic equilibrium between Gactin and F-actin is essential for maintaining cellular structure and responding to extracellular signals.In the context of microglia, actin dynamics play a critical role in determining their functional state and responsiveness.Actin's versatility allows microglia to rapidly remodel their cytoskeleton, facilitating processes such as phagocytosis, migration, and the formation of cellular protrusions necessary for nanoscale surveillance of the brain environment (Bernier et al., 2019).
In resting microglia, F-actin is mainly found in their cellular processes (Fig. 1B), facilitating efficient surveillance and response to the brain's environment (Bernier et al., 2019).This specific localization of F-actin propels the formation of filopodia, slender and highly dynamic structures crucial for microglia's sensing capacity and motility functions (Bernier et al., 2019).However, when microglia become activated, F-actin is redistributed throughout the cell body and localizes to a great extent to membrane ruffles (Fig. 1B).This shift in F-actin distribution reflects a significant change in microglial behavior from extensive surveillance to localized, small-scale movements (Bernier et al., 2019).Thus, activation induces a less ramified microglial phenotype, which mediates the microglial ability to transit from large-scale sensing to more localized responses (Bernier et al., 2019).
The role of actin in microglial function extends beyond the traditional concepts of cell scaffolds and cytoarchitecture.It is deeply involved in context-dependent responses of microglia to their environment.The emerging insights into the regulation of actin dynamics by molecules like Rho GTPases, such as RhoA, Rac1, and Cdc42, further underline the complexity of actin-mediated microglial functions.These regulators play essential and distinct roles in maintaining a delicate balance between the formation and disassembly of actin structures, allowing microglia to adapt their morphology and function in response to various stimuli and activation states.
For instance, RhoA, a Rho GTPase subfamily member critically Fig. 1.Correlation between microglial functional states and cytoskeleton components.The illustration presents an in-depth comparative analysis of microglial cells across three distinct functional states: Homeostatic (Resting), Activated, and Aged, with a focus on Cell Shape, F-actin, and Tubulin distribution and structure.A -Cell Shape: In the Homeostatic state, microglia display a highly branched, ramified structure, indicative of a stable, surveillance-centric state essential for continuous environmental monitoring.The Activated state is marked by a noticeable retraction of processes, denoting a shift from steady surveillance to a more dynamic state for rapid response to stimuli.The Aged state shows a simplified, deramified form, illustrating the impact of aging on cellular function and structure, often associated with a decline in surveillance capability.B -F-actin: In the resting state, F-actin predominantly localizes within cellular processes.This arrangement facilitates efficient surveillance and response to environmental stimuli in the brain.Upon activation, there is a notable redistribution of F-actin throughout the cell body, particularly at membrane ruffles.This shift signifies a transition from extensive surveillance to localized movements, reflecting a fundamental change in microglial behavior.As microglia age, a decline in profilin1 expression may lead to a reduction in F-actin content, mirroring the age-related changes in microglial dynamics and potentially altering their neuroprotective and inflammatory responses.C -Tubulin: In resting microglia, MTs are nucleated at Golgi outposts, maintaining a specific, stable morphology.Activation triggers significant MT remodeling, transitioning from a non-centrosomal parallel organization to a radial array anchored at the pericentrosomal material near the nucleus.This restructuring can be induced by stimuli like LPS-IFNγ or IL-4.Aging in microglia is associated with notable changes in MT stability, likely due to alterations in post-translational modifications and variations in the expression of tubulins and MAPs, leading to a disrupted MT network.
involved in actin remodeling, plays a pivotal role in the microglial functional state (Socodato et al., 2020).RhoA functions as a binary switch, cycling between a GDP-bound inactive state and a GTP-bound active state, thereby interacting with downstream effectors that potentially regulate various aspects of the microglial cytoskeleton and function.The ablation of RhoA in microglia is associated with amyloidosis, synapse loss, and memory deficits (Socodato et al., 2020).Complementing this view, Melo et al. identified Myh9 and Myh10,classical RhoA effectors in cytoskeleton remodeling, as critical regulators of microglial shape and function (Melo et al., 2021).Interestingly, Melo et al. also focus on cofilin, discussing its role in maintaining actin turnover (Melo et al., 2021), which might be crucial for regulating microglial protrusion extension.Further expanding our understanding, Bernier et al. identify intracellular cAMP as a dual regulator of microglial morphology (Bernier et al., 2019).Using elegant two-photon imaging, the authors show that cAMP promotes the growth of filopodia-like structures in microglia while simultaneously inducing microglial process retraction (Bernier et al., 2019), suggesting its role as a molecular switch in actin dynamics, likely complementary to RhoA.
The roles of other Rho GTPases like Rac1 and Cdc42 add complexity to the picture.Rac1 is a critical player in actin polymerization and lamellipodia formation, a mesh of branched actin networks crucial for driving microglia process ramification and motility (Socodato et al., 2023a).On the other hand, Cdc42 is typically associated with filopodia formation and might work in tandem with cAMP signaling to refine the surveillance capabilities of microglial cells.
Expanding on the interplay between Rac1 and Cdc42, it is worth noting that both have a shared mechanism that can modulate microglia's behavior through their interaction with the Arp2/3 complex.In microglia, the Arp2/3 complex is far from a mere bystander; it is wellknown as a critical facilitator in actin branching, vital for microglia's intricate tree-like morphology (James et al., 2020).Rac1, in particular, influences the Arp2/3 complex by interacting with the WAVE regulatory complex (WRC), a conglomerate of factors that includes Cyfip1, a protein associated with autism and intellectual disability.Cyfip1 is a crucial link between Rac1 and the Arp2/3 complex, facilitating the transmission of signals from Rac1 to activate the Arp2/3-dependent actin branching (James et al., 2020).This Rac1-WRC-Arp2/3 signaling axis is vital for the surveillance behavior of microglia, as evidenced by studies using Cyfip1 conditional knockout mouse models (James et al., 2020) and human iPSC-derived microglia (Sheridan et al., 2023).The loss of Cyfip1 leads to a reduction in microglial surveillance and phagocytic capacity (James et al., 2020;Sheridan et al., 2023), underscoring the importance of this pathway in both normal microglial function and its potential implications in neurodevelopmental disorders.

Microtubules: a new dimension in microglial dynamics and function
Microtubules (MTs), a fundamental component of the cellular cytoskeleton, are dynamic structures composed of α/β-tubulin heterodimers, playing diverse roles in cell shape maintenance, intracellular transport, and cell division (McKenna et al., 2023).The stability of MTs is dynamically regulated, often influenced by post-translational modifications, with acetylation on lysine 40 of α-tubulin being a notable example (Bär et al., 2022).This acetylation has been linked to enhanced stability and mechanical resistance of MTs, though it typically marks already stabilized structures rather than actively stabilizes them (Bär et al., 2022).The continual balance between growth and shrinkage of MTs is critical for their functional adaptability.γ-tubulin, while not a direct component of the MTs like αand β-tubulin, plays an essential role in the MT network.It is primarily located at microtubule-organizing centers (MOCs), where it is instrumental in initiating the assembly of MTs and setting their directional polarity.This is particularly relevant in microglia, where MTs are significantly remodeled as these cells transition between resting and reactive states (Adrian et al., 2023;Rosito et al., 2023).Key insights from Rosito et al.
(2023) and Adrian et al. (2023) have elucidated the intricate mechanisms behind this transformation, revealing how MT dynamics are integral to microglial function (Adrian et al., 2023;Rosito et al., 2023).In a resting state, MTs are nucleated at Golgi outposts (Fig. 1C) and exhibit a specific, stable morphology (Rosito et al., 2023).However, upon activation by stimuli such as lipopolysaccharide-interferon-γ (LPS-IFNγ) or interleukin-4 (IL-4), these MTs undergo significant remodeling (Fig. 1C), transitioning from a non-centrosomal parallel organization to a radial array anchored to pericentrosomal material (PCM) near the nucleus (Rosito et al., 2023).Rosito et al. (2023) and Adrian et al. (2023) studies employ advanced super-resolution microscopy to reveal the functional dynamics of MT reorganization.Rosito et al. (2023) demonstrate that γ-tubulin, a major MT nucleator, redistributes from a diffuse pattern in homeostatic cells to a concentrated pericentrosomal area in activated microglia.This redistribution depends on dynamic MTs and is rate-limiting for cytokine release, including IL-1b (Rosito et al., 2023).Adrian et al. (2023) corroborate this by elucidating the role of Cdk1 in cytokine trafficking and secretion, emphasizing that Cdk1 activation is essential for MT remodeling and functional cytokine release (Rosito et al., 2023).
However, the MT reorganization in activated microglia, particularly regarding stability, presents a complex picture.While Adrian et al. (2023), report a stable, centrosomally anchored MT array in LPS-treated microglia, this observation contrasts with the findings of Rosito et al. (2023).Rosito and colleagues noted a decrease in tubulin post-translational modifications, traditionally a proxy for MT stability, upon activation and recentering of the MT array.This discrepancy could be attributed to differences in the microglial activation protocols or the cellular states being examined.
Supporting this notion, Ilschner and Brandt also reported a decrease in MT stability in ameboid microglia (Ilschner and Brandt, 1996), further corroborating the observation of Rosito et al. that ameboid microglia exhibit higher MT plus end dynamicity.This evidence collectively indicates a possible decrease, rather than an increase, in MT stabilization during certain microglia activation states.Such variability in MT dynamics underscores the importance of further research to resolve these conflicting observations and fully understand the remodeling of the microglial MT cytoskeleton.
Adding another layer of complexity, Rosito et al. (2023) delve into the role of post-translational modifications, such as acetylation, in the context of MT dynamics within microglia.They observe that reactive microglia exhibit MT organizations that are less dense compared to resting microglia in vitro.In these cells, MTs extend throughout the cytoplasm, and while acetylation at the MT Organizing Center (MTOC) is a notable modification, this acetylation may not be primarily a stabilizing factor.Instead, it may accumulate on MTs that have already been stabilized through other mechanisms.This scenario points to a more complex network of biochemical modifications at play regulating MT stability and dynamics in microglia.
Regarding in vivo relevance, both works validate their findings in a more physiological context.Rosito et al. (2023) confirm that the pericentrosomal localization of γ-tubulin is a bona fide feature of activated microglia in an ex vivo paradigm.Adrian et al. (2023) validate the physiological relevance of Cdk1 activation using acute brain slices from Cx3CR1-GFP mice, showing that Cdk1 activation is required for the morphological changes of microglia in their endogenous environment.
In summary, these studies provide compelling evidence that remodeling the MT cytoskeleton, orchestrated by key molecular players like γ-tubulin and Cdk1, is critical for microglial transition from a homeostatic to a reactive state.They elucidate the molecular intricacies involved, such as the role of specific proteins and PCM in MT nucleation and the impact of MT dynamics on cytokine release.

Microtubules vs. actin: complementary or intertwined roles?
The traditional understanding of microglial reactivity often R. Socodato and J.B. Relvas categorizes actin and MTs into distinct functional realms.Actin is typically seen as critical for immediate, localized responses such as phagocytosis and motility.MTs are more associated with sustaining long-term cellular changes, encompassing cell division and intracellular transport.However, emerging studies highlighting a more integrated relationship between these two cytoskeletal components increasingly challenge this compartmentalized view.
This integration is orchestrated by key regulatory molecules such as the Rho GTPases, mainly RhoA, Rac1, and Cdc42, which are now understood to influence both actin and MT dynamics (Dogterom and Koenderink, 2019;Seetharaman and Etienne-Manneville, 2020).These small GTPases, traditionally recognized for their roles in actin modulation, are also pivotal in regulating MT stability and dynamics, thereby serving as crucial links in the crosstalk between actin and MTs (Dogterom and Koenderink, 2019; Seetharaman and Etienne-Manneville, 2020).Their roles extend beyond the singular modulation of either cytoskeletal system, emphasizing a coordinated regulatory mechanism that facilitates the interplay between actin and MTs.
Recent findings indicate that actin's role is not confined to rapid, localized responses.For instance, RhoA, pivotal for actin dynamics, has been implicated in long-term microglial changes that lead to neurodegeneration (Socodato et al., 2020) and metabolic reprogramming during neuroinflammation (Socodato et al., 2023b).Similarly, type-II myosins like Myh9 and Myh10 have differential roles in controlling microglial shape and function.Myh9 regulates cortical tension levels and affects microglia protrusion formation, while Myh10 is implicated in microglia inflammatory activation (Melo et al., 2021).The role of cAMP in regulating microglial morphology (Bernier et al., 2019) further complicates things.cAMP is a well-known secondary messenger in cellular signaling that influences the dynamics of both actin and MT.This suggests that intracellular signaling pathways may coordinate the activities of these two cytoskeletal elements during different functional states of microglia.These findings add another layer of complexity to the actin-MTs interplay, suggesting that actin dynamics contribute to long-term cellular changes, much like MTs.
Delving deeper into the molecular intricacies, the actin-MT interactions are mediated by various proteins that serve as signaling integrators like Rho GTPases and crosslinkers, such as the large multidomain proteins that bind to F-actin and MTs (Dogterom and Koenderink, 2019;Seetharaman and Etienne-Manneville, 2020).These proteins, along with MT plus-end-binding proteins, facilitate dynamic links between the growing MT plus ends and actin bundles, influencing the directionality and growth of MTs (Dogterom and Koenderink, 2019;Seetharaman and Etienne-Manneville, 2020).Additionally, through myosin motor activity, the actin cortex generates tension that affects cell shape and the positioning of the MT spindles.This is further exemplified by the role of formins and WASP family proteins in actin nucleation, which can be localized by MT ends, thereby integrating the dynamics of both cytoskeletal elements (Dogterom and Koenderink, 2019;Seetharaman and Etienne-Manneville, 2020).
Building on this, the crosstalk between actin and MTs extends to regulating MT dynamics by actin-associated proteins.For example, stabilizing MT ends by actin networks involves proteins that suppress MT dynamics, leading to stable yet adaptable linkages (Dogterom and Koenderink, 2019;Seetharaman and Etienne-Manneville, 2020).This is not a one-way interaction; MT can influence actin dynamics by localizing factors that promote actin polymerization (Dogterom and Koenderink, 2019;Seetharaman and Etienne-Manneville, 2020).The interplay is a complex regulatory network where proteins such as CLIP170 and mDia1 (a classical RhoA/Rac1 effector), associated with MT plus ends, can stimulate actin assembly, indicating a reciprocal regulatory mechanism (Dogterom and Koenderink, 2019;Seetharaman and Etienne-Manneville, 2020).Hence, the dynamic properties of actin and MTs are co-regulated, affecting immediate responses and long-term cellular states.

Cytoskeleton dysfunction: a proxy for microglial aging
Microglia undergo significant changes in morphology, motility, and function as the brain ages.These changes are likely contributed by alterations in microglia's actin and MT networks (Fig. 1A-C).Such modifications can impact their behavior, potentially leading to altered inflammatory responses and a shift in their neuroprotective roles in the aged brain.

Actin dynamics, microglia, and aging
Actin dynamics play a crucial role in microglia, and profilin-1 and cofilin are two important proteins involved in this process.Profilin-1 helps incorporate actin monomers (G-actin) into actin filaments (Factin), which is necessary for maintaining actin filament turnover.This function is crucial for microglial activities such as phagocytosis and motility.In aging microglia, these dynamics are disrupted, resulting in decreased motility and reduced ability to remodel their shape in response to external cues (Damani et al., 2011).The decline in the expression of profilin1 in aged microglia (Galatro et al., 2017) may lead to an overall decrease in F-actin content (Fig. 1B), which parallels the age-related changes in microglial dynamics.
Cofilin, on the other hand, severs and depolymerizes actin filaments.The activity of cofilin is regulated by phosphorylation, which inactivates it, while dephosphorylation by phosphatases activates it, enabling the severing of actin filaments and facilitating actin turnover.Cofilin's role in actin dynamics is further nuanced by its regulation through upstream modulators like Rac1 and signaling molecules such as cAMP.This complex regulation highlights the intricacy of actin dynamics in aging microglia.
Age-related neurodegenerative diseases, such as Parkinson's Disease (PD) and Dementia with Lewy Bodies (DLB), are characterized by the accumulation of protein aggregates, specifically α-synuclein (α-Syn), in neuronal cells.Recent research has provided valuable insights into an actin-related role contributing to the spread of these toxic aggregates (Chakraborty et al., 2023;Scheiblich et al., 2021).Chakraborty et al. examined the role of tunneling nanotubes (TNTs) in facilitating the bi-directional transfer of α-Syn and mitochondria between neuronal and microglial cells.Meanwhile, Scheiblich et al. explored how microglia engage in the degradation of α-Syn through TNTs.TNTs are F-actin-rich membrane protrusions that connect cells over long distances and have been implicated in neurodegenerative diseases.Cells can transfer cellular materials, including ions, organelles, and protein aggregates like α-Syn, through TNTs.Both studies suggest that microglia and TNTs work together to manage the spread of α-Syn (Chakraborty et al., 2023;Scheiblich et al., 2021).Chakraborty et al. suggest a mechanism to relieve the burden of these aggregates by revealing a bias in the transfer of α-Syn from neuronal cells to microglia.Scheiblich et al. complement this by dissecting how microglia use TNTs to engage in the degradation of α-Syn.Understanding the role of actin-related structures, such as TNTs, in the spread and degradation of α-Syn aggregates may provide new avenues for therapeutic interventions to increase the clearance of α-Syn and reduce protein accumulation in PD and DLB.
Understanding the actin-related molecular landscape in microglial function is critical for identifying potential therapeutic targets.The regulation of profilin-1 and cofilin in microglia is highly coordinated, and changes in their expression levels can have implications for microglial function, aging, and neurodegenerative diseases such as PD and DLB.Further research unraveling these complex interactions and their impact on microglial function is essential for identifying potential therapeutic targets.

MT dynamics, microglia, and aging
As microglia age, they undergo significant changes in MT dynamics, which may manifest through alterations in post-translational modifications (PTMs) such as acetylation or detyrosination, variations in tubulin isoforms, and the regulation of microtubule-associated proteins (MAPs).These changes could result in a reorganized MT network within aging microglia (Fig. 1C).It is plausible to consider that these modifications are closely linked to the aging process itself, indicating a complex relationship between MT dynamics and the development of agerelated neurodegenerative diseases.
In considering the impact of aging on MT stability, it is essential to explore two contrasting perspectives.On the one hand, aging might reduce MT stability due to changes in PTMs and MAP expression (Fig. 1C thinner green lines), which could impair microglial function and structural integrity.On the other hand, aging may conversely lead to increased MT stability and tubulin content (Fig. 1C thicker green lines).This increased stability could have distinct effects on microglial functionality.For instance, studies like those by Rosito et al. have shown that the application of taxol, an MT-stabilizing agent, can inhibit IL-1b upregulation and microglial reactivity, suggesting that a higher degree of MT stability might play a role in age-dependent alterations in microglial activation.
Furthermore, the age-related changes in the expression or activity of proteins such as stathmin (Chen et al., 2003;Carrette et al., 2006), known for their role in MT depolymerization, should be reconsidered within the context of microglial aging.Traditionally associated with contributing to MT instability and potentially adverse effects on microglial function during aging, the increased MT stability associated with aging might necessitate adjustments in stathmin expression or activity to maintain a balance essential for optimal microglial functionality.In light of the absence of definitive experimental evidence to support one scenario over the other in the context of aging, it is reasonable to consider both possibilities.
It is possible that changes in MT dynamics in aging microglia can also affect their interaction with other brain cells.For instance, disruptions in MT networks can impact the formation and maintenance of microglial processes that reach out to and retract from synapses, which can affect synaptic pruning and potentially contribute to synaptic dysfunction associated with aging.Another possibility is that altered MT stability can impact the microglia's immune response.This can reduce the microglia's ability to move towards and effectively eliminate debris or pathological protein aggregates.This inefficiency can contribute to the accumulation of such aggregates, creating a vicious cycle that further worsens neurodegenerative pathology.Lastly, modifying MT stability and function can help preserve or restore microglial health, thus mitigating the effects of aging on brain function.For example, drugs that stabilize MTs or regulate MAPs' activity can offer novel strategies to enhance microglial resilience against aging-related changes.
The complex molecular landscape of the microglial cytoskeleton, encompassing both the actin and MTs, reveals an intricate network of regulatory mechanisms potentially deeply impacted by aging.Key molecular players such as profilin-1, cofilin, MAPs, and Rho GTPases are central to these dynamics.Their roles might go beyond being mere spectators; they likely actively shape the aging process in microglial cells.This understanding shifts our perspective, suggesting that cytoskeletal dynamics in microglia are not merely consequences but fundamental drivers of the aging process.Thus, cytoskeletal dynamics serves as a valuable proxy for understanding and assessing microglial aging.It provides essential insights into the cellular mechanisms underlying age-related changes in the brain and highlights potential therapeutic targets.

Conclusion and future directions
The recent shift in focus toward the role of MTs in microglial reactivity has significantly impacted our understanding of microglial dynamics and function.While the importance of actin remains, identifying specific regulators like Cdk-1 and mechanisms such as centrosomal nucleation adds new dimensions that could be exploited for therapeutic interventions in brain diseases.However, much of this new understanding comes from in vitro studies, and complementary approaches, such as animal studies employing intravital imaging of both actin and MT cytoskeletons, are essential to validate these findings in more physiological contexts.Although these studies may present technical challenges, they are indispensable for confirming the dynamic patterns observed in recent studies.Successfully bridging the gap between in vitro and in vivo data will not only fortify our grasp of microglial cytoskeletal dynamics but also facilitate the development of targeted therapeutic strategies.
Understanding the mechanisms regulating actin and MTs interplay also opens up new avenues for research.Questions about their interaction at the molecular level during different stages of microglial activation remain.Furthermore, the specific factors and pathways governing the organization and function of both cytoskeleton components during microglial reactivity still need to be determined.These unanswered questions, which extend to the role of intracellular second messengers like cAMP, warrant further research.
Overall, ongoing research on MT and actin regulators in microglial reactivity challenges conventional paradigms and provides new insights.As we explore the intricacies of cytoskeletal dynamics in microglia, we are getting closer to a more complete understanding of these processes, which could have important implications for treating age-related neurological disorders.

Declaration of Competing Interest
The authors declare no competing interests related to this manuscript.