Distinct dynein complexes defined by DYNLRB1 and DYNLRB2 regulate mitotic and male meiotic spindle bipolarity

**Background:** Cytoplasmic dynein 1 is a microtubule motor complex essential for retrograde transport and cell division. While dynein's roles in mitosis are well-characterized, its meiosis-specific functions and subunit composition remain poorly understood. The light chain subunits are the most divergent class, with six paralogs in mammals, but their distinct roles are not fully defined. Male meiosis relies on the canonical centrosome system for spindle formation, distinct from acentrosomal oocyte meiosis, but specific regulatory mechanisms were unknown.

**Methods:** The authors generated Dynlrb2 knockout mice using a gene trap allele on a C57BL/6N background. They performed histological analysis, immunostaining of spermatocytes (using squash and chromosome spread techniques), immunoprecipitation from testis extracts, sucrose density gradient centrifugation, yeast two-hybrid assays, and single-cell RNA sequencing analysis of published mouse testis data. For mitotic studies, Dynlrb1 was knocked down using siRNA in B16-F1 mouse melanoma cells and HEK293 human cells. Rescue experiments were performed with RNAi-resistant constructs. Single-molecule imaging of purified GFP-dynein complexes was conducted in HeLa cells stably expressing GFP-labeled dynein HC, transfected with FLAG-tagged hDYNLRB1 or hDYNLRB2.

**Key Results:** DYNLRB2 protein was expressed exclusively in testis among adult mouse tissues, while its paralog DYNLRB1 was ubiquitously expressed. Single-cell RNA sequencing showed mutually exclusive expression: Dynlrb1 in mitotic cells (Sertoli cells, Leydig cells, spermatogonia) and Dynlrb2 in meiotic cells. DYNLRB2 co-immunoprecipitated with dynein intermediate chains and co-fractionated with other dynein subunits in high molecular weight fractions on sucrose gradients. DYNLRB2 localized to spindle poles and kinetochores in metaphase I and II spermatocytes.

Dynlrb2 knockout male mice were completely infertile with significantly smaller testes. Histological analysis showed no spermatozoa in the epididymis (0% of 3828 tubule sections vs 99.8% of 5114 in WT). TUNEL assays detected apoptosis specifically in metaphase I spermatocytes at stage XII seminiferous tubules. Chromosome spreads showed significant accumulation of metaphase I spermatocytes in knockout testes. The majority of Dynlrb2−/− metaphase I spermatocytes formed multipolar spindles with fragmented pericentriolar material (PCM). Astral microtubules were completely depleted. Chromosomes were largely misaligned, and the spindle assembly checkpoint was activated (significant BubR1 accumulation at kinetochores).

Two distinct mechanisms caused PCM fragmentation: (1) premature centriole disengagement—approximately 23% of Dynlrb2−/− metaphase I cells showed premature separation of mother and daughter centrioles, with loss of STIL and SAS-6 signals from daughter centrioles; (2) NuMA mislocalization—NuMA showed a 60% reduction in intensity at spindle poles in knockout spermatocytes compared to WT controls, and its symmetric distribution at poles was disrupted. Importantly, other dynein subunits (DYNC1H1, DYNLL1, p150) localized normally, and dynein complex formation was not impaired in the absence of DYNLRB2.

In mitotic B16-F1 cells, Dynlrb1 knockdown caused significant mitotic arrest with multipolar spindles and fragmented PCM. Unlike meiotic cells, centriole overduplication (more than 4 CETN2 foci) was the primary cause. NuMA signal intensity was reduced to 62% of control levels. These defects were recapitulated in human HEK293 cells. Ectopic expression of DYNLRB2 fully rescued the multipolarity caused by Dynlrb1 knockdown. Single-molecule analysis showed that both DYNLRB1- and DYNLRB2-containing dynein complexes moved processively along microtubules with comparable velocities.

**Clinical Implications:** This study reveals that distinct dynein complexes containing DYNLRB1 or DYNLRB2 are separately used in mitotic and meiotic spindle formation, with NuMA as a common target. The findings provide mechanistic insight into male infertility caused by meiotic arrest, identifying DYNLRB2 as a potential target for understanding non-obstructive azoospermia. The demonstration that DYNLRB2-containing dynein complexes ensure robust NuMA accumulation at spindle poles—more strongly than DYNLRB1—suggests that male meiotic cells require specialized machinery to handle the larger bivalent chromosomes. The study also resolves a long-standing controversy about whether NuMA localization at spindle poles depends on dynein, showing that it does, through DYNLRB1/2-containing complexes.