Entropic swelling bursts the nuclear envelope

Besides containing the genetic information of chromosomes, the cell nucleus is a very dynamic organelle. Its morphology can vary from cell type to cell type with pronounced associations with cell functions and gene expression. In addition, it is flexible, depending on the cell environment and cell activity [95]. From a biomechanical point of view, the nucleus

CHAPTER 4 - Discussion and outlook

Dissertation - Elsa Neubert

pointed out, the neutrophil nucleus has to allow massive structural alterations to squeeze through the endothelium into the tissue. The description of NETosis adds several new requirements for its architecture.

The neutrophil nucleus is not only characterized by its eponymous lobulated shape, but also by a unique composition of its envelope. These characteristics develop during granulopoiesis and comprise the lack in lamin A/C and proteins of the LINC complex, decreased lamin B levels as well as markedly increased LBR. These changes most likely contribute to the remarkable flexibility of neutrophils for instance during migration [94, 133]. Interestingly, the absence of lamin A/C does not only mediate overall flexibility but also causes a lack of lamin A/C-binding to chromatin [379] and makes the cell softer and less resistant as shown in migration studies [380]. Together with the decreased expression of the tethering LINC complex [119], it possibly allows the chromatin to redistribute more easily and faster as recently discussed by Manley et al. [94, 237, 381]. Additionally, the rapid re- and- decondensation of chromatin in neutrophils reported under hypotonic conditions supported this hypothesis [100]. As a consequence, the nuclear envelope is overall more fragile. This makes neutrophils evolutionarily prepared for profound reorganizations and most likely allows the fast chromatin remodeling observed in the first phase of NETosis (P1).

Subsequently, it prepares the following chromatin decondensation in P2 (manuscript I, Fig. 1 and 2). It is likely that the nuclear envelope increases its “susceptibility to chromatin-exerted pressure” [94]. Interestingly, murine neutrophils and HL-60 model cells contain more lamins and, at the same time, appear less fragile [99, 382]. It is therefore likely that these cells are less predisposed for chromatin remodeling as well as nuclear envelope rupture, which may lead to a delayed onset of P2. Indeed, stimulation of murine neutrophils yielded lower NET rates than human neutrophils, as repeatedly reported [383] and verified in manuscript II (Fig. 1 vs. Fig. 3). It would be interesting to study this phenomenon in more detail in our phase model to clarify whether NETosis in murine neutrophils shows a prolonged P1 and therefore reaches the point of no return later, or whether mice generally have lower NET rates for other reasons.

In their review, Manley et al. also correlated the lack in lamin A/C with the short life-span of neutrophils, as the lack of lamin A/C renders the nucleus less protected and more prone to cell death [384] and DNA damage [385, 386]. This is of particular interest for aging neutrophils, which show an activated, pro-inflammatory phenotype [71, 73] including higher NET rates [387]. It is possible that DNA alterations can prime chromatin decondensation and facilitate the onset of chromatin swelling. In addition, NE from aging neutrophils can be released more easily from granules, cleave gasdermin D and induce lytic cell death by pore-formation in membranes [388], a mechanism which was only recently implicated in NETosis.

This could affect the integrity of membranes and thereby prime the cells for NETosis.

In contrast, neutrophils from older hosts display impaired NETosis [76, 77]. Interestingly, alterations in lamin proteins were previously described for different cell types of aging hosts [389, 390]. Whether neutrophils also show these changes in lamin expression, such as increase in lamin A/C levels, and whether these alterations correlate with impaired NETosis

In addition to its unique composition, the nuclear envelope can become modified further during NET formation, for instance by phosphorylation of the remaining lamin A/C [235].

Based on our phase model (manuscript I), it is expected that these modifications take place predominantly in the first and active phase of NETosis (P1) (manuscript I, Fig. 3). Whether this particular alteration has a functional implication in the subsequent envelope rupture or whether it is a by-stander product of the mitosis pathway is unclear. However, it is likely that the nuclear envelope undergoes even more profound changes than anticipated. For instance, it was postulated that the passive or active decrease/down-regulation of LBR, the most crucial tethering molecule in the neutrophil nuclear envelope, supports delobulation and decreases heterochromatin detachment from the nuclear membrane [94]. Similar mechanisms are likely to take place during P1. LBR decrease could mainly support chromatin remodeling and the onset of chromatin swelling. Nonetheless, this hypothesis requires further investigation especially in neutrophils from mice lacking LBR [391] or time-resolved observations of the nuclear envelope proteins correlated with the state of chromatin decondensation in human neutrophils.

In two independent studies, Fuchs et al. and Amulic et al., postulated nuclear membrane dissolution during NETosis into vesicles [98, 235]. As already discussed in manuscript I, this process coincides with time-points of our P2, clearly after the start of chromatin decondensation. At which stage of NETosis the nuclear membrane actually starts to disintegrate was not further addressed in these two publications. In our study, we observed the rupture of the lamina (lamin B1) significantly earlier, exactly with the onset of P2 (t1) (manuscript I, Fig. 2). Together with our real-time observations, which show an impressive increase in chromatin area directly after t1, it is very likely that the generated entropic pressure of the swelling chromatin is sufficient to burst at least the nuclear lamina and literally ‘free’ the chromatin to swell further within the cytoplasm. Whether this corresponds with the burst of the nuclear membrane, or whether the membrane is already significantly weakened beforehand and starts to dissolve already within P1, however, is not clear.

Potential mechanisms of membrane weakening include gasdermin D-induced pore formation [229, 230] and alterations of the supporting cytoskeleton [228]. Most likely, the membrane is prepared by specific modifications for rupture by the expanding chromatin.

These considerations are even more important given that the release of ETs is not restricted to human neutrophils, but rather represents a highly conserved process among different species, cells and organisms. Therefore, the fluidity of chromatin within the neutrophil nucleus and the unique composition of its nuclear envelope might prime neutrophils, especially human neutrophils, to undergo NETosis. However, this is most likely not the only underlying mechanism. It is expected that cells either undergo a conserved program to prepare the nuclear envelope for rupture by swelling chromatin, or the entropic chromatin swelling itself, enabled by degradation and modification of DNA and histones, is sufficient to

‘burst’ the nuclear envelope regardless of its predisposition. Further live-cell observations, however, are needed to correlate the state of chromatin remodeling in the first phase with the exact composition of the nuclear envelope. Likewise, studies on isolated nuclei from

CHAPTER 4 - Discussion and outlook

Dissertation - Elsa Neubert

different species could clarify how much pressure is generated by the swelling chromatin and whether this pressure suffices to burst the nuclear envelope of nuclei from all species.