Microtubules and Dynein Regulate Human Neutrophil Nuclear Volume and Hypersegmentation During H. pylori Infection

Abstract

Neutrophils (also called polymorphonuclear leukocytes, PMNs) are heterogeneous and can exhibit considerable phenotypic and functional plasticity. In keeping with this, we discovered previously that Helicobacter pylori infection induces N1-like subtype differentiation of human PMNs that is notable for profound nuclear hypersegmentation. Herein, we utilized biochemical approaches and confocal and super-resolution microscopy to gain insight into the underlying molecular mechanisms. Sensitivity to inhibition by nocodazole and taxol indicated that microtubule dynamics were required to induce and sustain hypersegmentation, and super-resolution Stimulated Emission Depletion (STED) imaging demonstrated that microtubules were significantly more abundant and longer in hypersegmented cells. Dynein activity was also required, and enrichment of this motor protein at the nuclear periphery was enhanced following H. pylori infection. In contrast, centrosome splitting did not occur, and lamin B receptor abundance and ER morphology were unchanged. Finally, analysis of STED image stacks using Imaris software revealed that nuclear volume increased markedly prior to the onset of hypersegmentation and that nuclear size was differentially modulated by nocodazole and taxol in the presence and absence of infection. Taken together, our data define a new mechanism of hypersegmentation that is mediated by microtubules and dynein and as such advance understanding of processes that regulate nuclear morphology.

Helicobacter pylori-infected human neutrophils exhibit impaired chemotaxis and a uropod retraction defect

“…For infection, PMNs were suspended at a concentration of 1 x 10 6 /mL and H. pylori were added at a 5:1 multiplicity of infection (MOI) ( 29 , 35 ). Cultures of 1-2 mL each in snap-cap polypropylene tubes were incubated at 37°C under humidified microaerophilic conditions for the amounts of time indicated below.…”

“…( 42 ), and then blocked overnight as described above. To detect microtubules (MTs) cells were fixed for 15 min in 2% PFA/0.1% glutaraldehyde in and then permeabilized with 0.5% SDS as we recently described ( 35 ) prior to blocking as described above.…”

Roles of the nucleus in leukocyte migration

“…Microtubules seem necessary for nuclear lobulation, as the formation of nuclear lobes is inhibited in nocodazole‐treated HL‐60 cells 91 . Nocodazole also inhibits nuclear hyperlobulation induced by Helicobacter pylori 92 . In addition, dynein becomes enriched on the nuclei and is involved in lobulation 92 .…”

“…Nocodazole also inhibits nuclear hyperlobulation induced by Helicobacter pylori 92 . In addition, dynein becomes enriched on the nuclei and is involved in lobulation 92 . Depletion of α‐dystrobrevin, a microtubule‐ and kinesin‐binding protein, also leads to rounded nuclei 93 .…”

“…Depletion of α‐dystrobrevin, a microtubule‐ and kinesin‐binding protein, also leads to rounded nuclei 93 . Interestingly, preexisting nuclear lobes are not affected by the loss of microtubules, 89,92 suggesting that microtubules are only involved in the formation, but not the maintenance of the nuclear lobes.…”