The ability to fluorescently label microtubules in live cells has enabled numerous studies of motile and mitotic processes. Such studies are particularly useful in budding yeast due to the ease with which they can be genetically manipulated and imaged by live cell fluorescence microscopy. Due to problems associated with fusing genes encoding fluorescent proteins (FPs) to the native a-tubulin (TUB1) gene, the FP-Tub1 fusion is generally integrated into the genome such that the endogenous TUB1 locus is left intact. Although such modifications have no apparent consequences on cell viability, it is unknown if these genome integrated FP-tubulin fusions negatively affect microtubule functions. Thus, a simple, economical, and highly sensitive assay of microtubule function is required. Furthermore, the current plasmids available for generation of FP-Tub1 fusions have not kept pace with the development of improved FPs. Here, we have developed a simple and sensitive assay of microtubule function that is sufficient to identify microtubule defects that were not apparent by fluorescence microscopy or cell growth assays. Using results obtained from this assay, we have engineered a new family of thirty FP-Tub1 plasmids that employ various improved FPs and numerous selectable markers that upon genome integration have no apparent defect on microtubule function.

Cortical dynein generates pulling forces via microtubule (MT) end capture-shrinkage and lateral MT sliding mechanisms. In Saccharomyces cerevisiae, the dynein attachment molecule Num1 interacts with endoplasmic reticulum (ER) and mitochondria to facilitate spindle positioning across the mother-bud neck, but direct evidence for how these cortical contacts regulate dynein-dependent pulling forces is lacking. We show that loss of Scs2/Scs22, ER tethering proteins, resulted in defective Num1 distribution and loss of dynein-dependent MT sliding, the hallmark of dynein function. Cells lacking Scs2/Scs22 performed spindle positioning via MT end capture-shrinkage mechanism, requiring dynein anchorage to an ER- and mitochondria-independent population of Num1, dynein motor activity, and CAP-Gly domain of dynactin Nip100/p150Glued subunit. Additionally, a CAAX-targeted Num1 rescued loss of lateral patches and MT sliding in the absence of Scs2/Scs22. These results reveal distinct populations of Num1 and underline the importance of their spatial distribution as a critical factor for regulating dynein pulling force.

Current model for spindle positioning requires attachment of the microtubule (MT) motor cytoplasmic dynein to the cell cortex, where it generates pulling force on astral MTs to effect spindle displacement. How dynein is anchored by cortical attachment machinery to generate large spindle-pulling forces remains unclear. Here, we show that cortical clustering of Num1, the yeast dynein attachment molecule, is limited by its assembly factor Mdm36. Overexpression of Mdm36 results in an overall enhancement of Num1 clustering but reveals a population of dim Num1 clusters that mediate dynein-anchoring at the cell cortex. Direct imaging shows that bud-localized, dim Num1 clusters containing only ∼6 copies of Num1 molecules mediate dynein-dependent spindle pulling via lateral MT sliding mechanism. Mutations affecting Num1 clustering interfere with mitochondrial tethering but not dynein-based spindle-pulling function of Num1. We propose that formation of small ensembles of attachment molecules is sufficient for dynein anchorage and cortical generation of large spindle-pulling force.

13 Current model for spindle positioning requires attachment of the microtubule (MT) motor 14 cytoplasmic dynein to the cell cortex, where it generates pulling force on astral MTs to effect 15 spindle displacement. How dynein is anchored by cortical attachment machinery to generate 16 large spindle-pulling forces remains unclear. Here, we show that cortical clustering of Num1, the 17 yeast dynein attachment molecule, is limited by Mdm36. Overexpression of Mdm36 results in an 18 overall enhancement of Num1 clustering but reveals a population of dim Num1 clusters that 19 mediate dynein-anchoring at the cell cortex. Direct imaging shows that bud-localized, dim Num1 20 clusters containing only ~6 copies of Num1 molecules mediate dynein-dependent spindle 21 pulling via lateral MT sliding mechanism. Mutations affecting Num1 clustering interfere with 22 mitochondrial tethering but not dynein-based spindle-pulling function of Num1. We propose that 23 formation of small ensembles of attachment molecules is sufficient for dynein anchorage and 24 cortical generation of large spindle-pulling force. 25 26 4 al. , 2010; Lackner et al., 2013). Although it is well-accepted that dynein exerts spindle pulling 53 force at cortical Num1 sites, the abundance and heterogeneity of Num1 patches along the cell 54 cortex (Heil-Chapdelaine et al., 2000; Omer et al., 2018; Schmit et al., 2018) has made it 55 impossible to follow the effects of astral MT plus end interaction with individual cortical Num1 56 sites, a prerequisite for understanding how clustering might impact dynein force amplification. 57 To our knowledge, contacts between astral MT plus end and individual Num1 foci have not 58 been observed for MT sliding, the in vivo hallmark of dynein-mediated spindle pulling (Adames 59 and Cooper, 2000; Yeh et al., 2000), hence the size of Num1 clusters required for this classic 60 dynein-dependent microtubule-cortex interaction remains unknown. Additionally, recent work61 shows that organelles such as mitochondria and endoplasmic reticulum (ER) are involved in 62 regulating Num1 cluster formation: a subset of cortical Num1 clusters appears to require 63 mitochondria for their assembly (Kraft and Lackner, 2017), whereas another population requires 64 the ER tethering proteins Scs2/Scs22 for their distribution throughout the cell cortex (Chao et 65 al., 2014; Omer et al., 2018). The general hypothesis emerging from these studies is that 66 distinct populations of Num1 clusters might exist at the cell periphery, but whether different 67 pools of Num1 could be performing different Num1 functions -namely, dynein anchoring and 68 mitochondrial tethering -remains a total mystery.69 Here, we set out to characterize the role of Mdm36 in Num1 clustering and found that, in 70 contrast to the prevailing notion for dynein-anchoring proteins, enhancing Num1 clustering 71 unexpectedly reduces dynein recruitment to the cell cortex, but without affecting dynein function 72 in spindle positioning. We report direct observation of MT sliding occurring upon...

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