In a recent study published in Cell Reports journal Dr. M. Rosito and her collaborators find that in microglia cells, the transition between homeostatic and reactive states is characterized by a dramatic reorganization of the microtubule cytoskeleton. In this study, the authors show that activated microglia provide an example of microtubule reorganization from a non-centrosomal array of parallel and stable microtubules to a radial array of more dynamic microtubules.
Among the numerous techniques used within this work, in this application note we report how CrestOptics spinning disk confocal (CF) and super-resolution (SR) systems were fundamental to studying microtubule remodeling and identifying defined ultrastructural elements typical of each microglial functional state. For a more complete view of all the brilliant results obtained in this work, please refer to the complete paper.
Microglia are the primary immune cells of the central nervous system. In their homeostatic state, they exhibit a ramified morphology and continuously patrol the local environment by extending and retracting their highly motile processes. During neuronal inflammation or after injury, microglia undergo dramatic changes in gene expression and morphology, displaying a characteristic ameboid shape typical of this activated state. Changes in cellular morphology and symmetry are associated with the extensive reorganization of both the actin and microtubule (MT) cytoskeletons and authors used both CF and SR microscopy to unveil that unique rearrangement of the MT cytoskeleton is required for the morphological changes during microglia transition from homeostatic to reactive states.
Confocal CF immunofluorescence analysis reveals a different MT orientation in homeostatic, activated and alternatively activated microglia.
To investigate the organization of the MT cytoskeleton in homeostatic and reactive microglia, the authors used primary mouse microglia cultures in which the presence of ramified cells (homeostatic microglia) was maintained by growth factors secreted by astrocytes. To orientate microglia toward different reactivity states, cells were challenged with lipopolysaccharide-interferon-g (LPS-IFNγ) for activated microglia or interleukin-4 (IL-4) for alternatively activated microglia.
Rosito et al. hypothesized that in microglia, the transition from the homeostatic phenotype to a migrating reactive state would be paralleled by prominent changes in cell polarity driven by the remodeling of MT anchoring and orientation. To evaluate this, they analyzed the localization and expression of MT plus-end end binding (EB, marker of actively growing MT plus ends) and minus-end CAMSAP (a protein involved in regulating the formation and stability of non-centrosomal MT arrays by capping free MT minus ends) markers in homeostatic, activated and alternatively activated microglia cells (Figure 1).
The results of CF immunofluorescence analysis indicated that EB1 decorated most free MT ends that extended toward the cell periphery in activated and alternatively activated microglia, confirming the presence of a prominent pool of dynamic MTs arranged radially with their minus ends attached to the perinuclear region. Although to a lesser extent, EB1 comets were also clearly visible at MT ends in homeostatic microglia. Furthermore, due to the high-resolution images obtained with the CrestOptics confocal spinning disk, analysis of fluorescence intensity gradients of EB-positive comets confirmed the presence of a population of retrograde comets in homeostatic microglia and alternatively activated microglia as opposed to activated microglia.
Figure 1: Representative immunofluorescence images of EB1 (magenta) and tyrosinated α-tubulin (Tyr tub) (green) in homeostatic (Homeo), activated (LPS-IFNγ) and alternatively activated (IL-4) microglia. Hoechst for nuclei visualization, blue. Scale bar, 20 um (5 um in zoom images).
Detection of a pool of retrograde comets in homeostatic microglia suggested the presence of non-centrosomal MT arrays which the authors investigated by analyzing the expression and distribution of endogenous CAMSAP2. While in homeostatic and alternatively activated cells CAMSAP2 was distributed as clustered puncta among cell ramifications (Figure 2 and 3, arrows), cytosolic CAMSAP2 staining was detected only around the perinuclear region in activated microglia.
Furthermore, the authors found that while endogenous CAMSAP2 was distributed as clustered puncta among cell ramifications in homeostatic and alternatively activated cells, cytosolic CAMSAP2 staining was only detected around the perinuclear region in activated microglia (Figures 2 and 3, arrows).
Figure 2: Representative Z-projection CF images showing CAMSAP2 (magenta) and Tyr tub (green) signal in Homeo-, LPS-IFNγ, or IL-4-treated microglia. Hoechst for nuclei visualization, blue. Scale bar, 20 um (5 um in zoom images).
Figure 3: Single CF planes at higher magnification of CAMSAP2 (magenta) and Tyr tub (green) signal in Homeo-, LPS-IFNγ-, or IL-4 treated microglia. Scale bar 5um.
These data indicate that both homeostatic and alternatively activated microglia exhibit mixed MT polarity patterns, and the acquisition of an activated phenotype is a unique example of remodeling the MT cytoskeleton from a parallel non-centrosomal to a radial array of MTs anchored to pericentrosomal microtubule organizing center (MTOCs) through their minus ends.
SR microscopy reveals pericentrosomal redistribution of microtubule-nucleating material in activated microglia.
The recruitment of pericentriolar material (PCM) represents a functional step during macrophage activation. Rosito et al. thus investigated whether the recruitment of γ-tubulin to a pericentrosomal area was also a hallmark of microglia activation.
In order to appreciate subcellular details and γ-tubulin subcellular distribution, the authors used a CrestOptics Structured Illumination Microscopy (SIM) system revealing that activated microglia displayed multiple γ-tubulin puncta localizing to a perinuclear region (Figure 4A). Moreover, the quantification of the number of γ-tubulin puncta indicated that activated cells had more than three puncta (Figure 4B); conversely, homeostatic and alternatively activated microglia displayed only one or two γ-tubulin puncta.
A
B
Figure 4: (A) Representative SR images showing γ-tubulin (γ-tub) puncta (magenta) and tyrosinated α-tubulin (left; Tyr Tub, green) immunolabeling in activated (LPS-IFNγ) microglia (middle). Hoechst for nuclei visualization, blue. Scale bar 5 um. Right: relative volume view (top) and 3D rendering (bottom) of γ-tub puncta (magenta) acquired via structured illumination microscopy. (B) Bar chart reporting the percentage of cells displaying 1-2 γ-tub puncta (white bars) or >3 γ-tub puncta (black bars) in homeostatic (Homeo), activated (LPS-IFNγ), or alternatively activated (IL-4) microglia. Values are expressed as mean ± SEM from 3 independent experiments.
As shown in Figure 4, the use of SIM enhances contrast and improves the resolution of subcellular details thus enabling the correct visualization of microglial cytoskeleton details and demonstrating the importance of maturation of pericentrosomal microtubule organizing centers in activated microglia.
Conclusions
Using CrestOptics Confocal CF and SR systems the authors were able to perform immunofluorescence analyses to study how microglia activation affects microtubule cytoskeleton remodeling.
It is shown that during classical and alternative activation, microglia MTs become less stable and more dynamic, suggesting that the acquisition of new cellular functions modulates MT stability.
Moreover, microglia activation results in enhanced γ-tubulin localization to puncta around the centrosome and de novo PCM and MTOC maturation, providing a distinct marker of microglia reactivity in live-imaging studies.
In conclusion, the combination of CrestOptics spinning disk CF and SIM represents a powerful solution to improve image resolution and obtain the cellular details necessary to study the cytoskeletal changes underlying the morphological and functional changes of microglia in different physiological and pathological contexts.
Methods
All the acquisitions presented in this Application Note were performed with CrestOptics spinning disk and SIM SR systems.
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Acknowledgments
This application note result from a collaboration between CrestOptics, Sapienza University of Rome, and the Center for Life Nano & NeuroScience.
Data presented in this Application Note are courtesy of Dr. Maria Rosito and Prof. Silvia Di Angelantonio as published in Rosito et al., 2023 and have been reused following approval from the authors. Re-use of images from this article is done under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND 4.0) license.
For a more complete view of all the brilliant results obtained in this work, please refer to the complete paper.
We want to acknowledge Dr. Maria Rosito, Prof. Silvia Di Angelantonio, Prof. Francesca Bartolini and Dr. Caterina Sanchini for their support in drafting this application note.
